U.S. patent application number 16/333333 was filed with the patent office on 2019-07-11 for nucleoside derivative or salt thereof, polynucleotide synthesis reagent, method for producing polynucleotide, polynucleotide, an.
This patent application is currently assigned to NEC Solution Innovators, Ltd.. The applicant listed for this patent is NATIONAL UNIVERSITY CORPORATION GUNMA UNIVERSITY, NEC Solution Innovators, Ltd.. Invention is credited to Jou AKITOMI, Katsunori HORII, Naoto KANEKO, Masayasu KUWAHARA, Hirotaka MINAGAWA, Iwao WAGA.
Application Number | 20190211048 16/333333 |
Document ID | / |
Family ID | 61619069 |
Filed Date | 2019-07-11 |
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United States Patent
Application |
20190211048 |
Kind Code |
A1 |
MINAGAWA; Hirotaka ; et
al. |
July 11, 2019 |
NUCLEOSIDE DERIVATIVE OR SALT THEREOF, POLYNUCLEOTIDE SYNTHESIS
REAGENT, METHOD FOR PRODUCING POLYNUCLEOTIDE, POLYNUCLEOTIDE, AND
METHOD FOR PRODUCING BINDING NUCLEIC ACID MOLECULE
Abstract
The present invention provides a novel nucleoside derivative or
a salt thereof, a polynucleotide synthesis reagent, a method for
producing a polynucleotide, a polynucleotide, and a method for
producing a binding nucleic acid molecule. The nucleoside
derivative or a salt thereof of the present invention is
represented by the following chemical formula (1): ##STR00001##
where in the chemical formula (1), Su is an atomic group having a
sugar skeleton at a nucleoside residue or an atomic group having a
sugar phosphate skeleton at a nucleotide residue, and may or may
not have a protecting group, L.sup.1 and L.sup.2 are each
independently a straight-chain or branched, saturated or
unsaturated hydrocarbon group having 2 to 10 carbon atoms, X.sup.1
and X.sup.2 are each independently an imino group (--NR.sup.1--),
an ether group (--O--), or a thioether group (--S--), and the
R.sup.1 is a hydrogen atom or a straight-chain or branched,
saturated or unsaturated hydrocarbon group having 2 to 10 carbon
atoms.
Inventors: |
MINAGAWA; Hirotaka; (Tokyo,
JP) ; HORII; Katsunori; (Tokyo, JP) ; AKITOMI;
Jou; (Tokyo, JP) ; KANEKO; Naoto; (Tokyo,
JP) ; WAGA; Iwao; (Tokyo, JP) ; KUWAHARA;
Masayasu; (Maebashi-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NEC Solution Innovators, Ltd.
NATIONAL UNIVERSITY CORPORATION GUNMA UNIVERSITY |
Tokyo
Maebashi-shi, Gunma |
|
JP
JP |
|
|
Assignee: |
NEC Solution Innovators,
Ltd.
Tokyo
JP
NATIONAL UNIVERSITY CORPORATION GUNMA UNIVERSITY
Maebashi-shi, Gunma
JP
|
Family ID: |
61619069 |
Appl. No.: |
16/333333 |
Filed: |
September 14, 2017 |
PCT Filed: |
September 14, 2017 |
PCT NO: |
PCT/JP2017/033211 |
371 Date: |
March 14, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/115 20130101;
C07K 16/00 20130101; C07H 21/04 20130101; C07H 21/02 20130101; C07H
19/14 20130101; Y02P 20/55 20151101; C07H 19/20 20130101 |
International
Class: |
C07H 19/20 20060101
C07H019/20; C07H 21/04 20060101 C07H021/04; C07H 21/02 20060101
C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 15, 2016 |
JP |
2016-180894 |
May 30, 2017 |
JP |
PCT/JP2017/020065 |
Claims
1. A nucleoside derivative or a salt thereof, represented by the
following chemical formula (1): ##STR00019## where in the chemical
formula (1), Su is an atomic group having a sugar skeleton at a
nucleoside residue or an atomic group having a sugar phosphate
skeleton at a nucleotide residue, and may or may not have a
protecting group, L.sup.1 and L.sup.2 are each independently a
straight-chain or branched, saturated or unsaturated hydrocarbon
group having 2 to 10 carbon atoms, X.sup.1 and X.sup.2 are each
independently an imino group (--NR.sup.1--), an ether group
(--O--), or a thioether group (--S--), and the R.sup.1 is a
hydrogen atom or a straight-chain or branched, saturated or
unsaturated hydrocarbon group having 2 to 10 carbon atoms.
2. The nucleoside derivative or a salt thereof according to claim
1, wherein the X.sup.1 is an imino group (--NR.sup.1--).
3. The nucleoside derivative or a salt thereof according to claim
1, wherein the X.sup.2 is an imino group (--NR.sup.1--).
4. The nucleoside derivative or a salt thereof according to claim
2, wherein the R.sup.1 is a hydrogen atom.
5. The nucleoside derivative or a salt thereof according to claim
1, wherein the L.sup.1 is a vinylene group (--CH.dbd.CH--).
6. The nucleoside derivative or a salt thereof according to claim
1, wherein the L.sup.2 is an ethylene group
(--CH.sub.2--CH.sub.2--).
7. The nucleoside derivative or a salt thereof according to claim
1, wherein the atomic group having a sugar skeleton at a nucleoside
residue or the atomic group having a sugar phosphate skeleton at a
nucleotide residue is represented by the following chemical formula
(2): ##STR00020## where in the chemical formula (2), R.sup.2 is a
hydrogen atom, a protecting group, or a group represented by the
following chemical formula (3), R.sup.3 is a hydrogen atom, a
protecting group, or a phosphoramidite group, R.sup.4 is a hydrogen
atom, a fluorine atom, a hydroxyl group, an amino group, or a
mercapto group, ##STR00021## where in the chemical formula (3), Y
is an oxygen atom or a sulfur atom, Z is a hydroxyl group or an
imidazole group, and m is an integer of 1 to 10.
8. The nucleoside derivative or a salt thereof according to claim
1, wherein the nucleoside derivative represented by the chemical
formula (1) is a nucleoside derivative represented by the following
chemical formula (4): ##STR00022##
9. A polynucleotide synthesis reagent comprising a nucleotide
derivative or a salt thereof that comprises the nucleoside
derivative or a salt thereof according to claim 1.
10. A method for producing a polynucleotide, comprising the step of
synthesizing a polynucleotide using a nucleotide derivative or a
salt thereof that comprises the nucleoside derivative or a salt
thereof according to claim 1.
11-15. (canceled)
Description
TECHNICAL FIELD
[0001] The present invention relates to a nucleoside derivative or
a salt thereof, a polynucleotide synthesis reagent, a method for
producing a polynucleotide, a polynucleotide, and a method for
producing a binding nucleic acid molecule.
BACKGROUND ART
[0002] In order to analyze a target in a specimen, a binding
molecule that binds to a target is used. In addition to an
antibody, a binding nucleic acid molecule that binds to a target
such as an aptamer is also used as a binding molecule that binds to
the target (Patent Literature 1).
[0003] As a method for obtaining the binding nucleic acid molecule,
a SELEX (Systematic Evolution of Ligands by Exponential Enrichment)
method in which a target is caused to come into contact with a
large number of candidate polynucleotides and a polynucleotide that
binds to the target among the candidate polynucleotides is selected
as the binding nucleic acid molecule is known. When a binding
nucleic acid molecule is obtained by the SELEX method, a modified
nucleoside molecule obtained by modifying a natural nucleoside
molecule is also used in addition to a natural nucleoside molecule
that constitutes the binding nucleic acid molecule.
[0004] However, with known natural nucleosides and derivatives
thereof, there are targets for which binding nucleic acid molecules
with sufficient binding ability cannot be obtained. Therefore,
there is a need for modified nucleoside derivatives that can be
used, for example, in the production of aptamers.
CITATION LIST
Patent Literature
[0005] Patent Literature 1: JP 2012-200204 A
SUMMARY OF INVENTION
Technical Problem
[0006] Hence, the present invention is intended to provide a novel
nucleoside derivative or a salt thereof, a polynucleotide synthesis
reagent, a method for producing a polynucleotide, a polynucleotide,
and a method for producing a binding nucleic acid molecule.
Solution to Problem
[0007] The nucleoside derivative or a salt thereof of the present
invention is represented by the following chemical formula (1).
##STR00002##
in the chemical formula (1), Su is an atomic group having a sugar
skeleton at a nucleoside residue or an atomic group having a sugar
phosphate skeleton at a nucleotide residue, and may or may not have
a protecting group, L.sup.1 and L.sup.2 are each independently a
straight-chain or branched, saturated or unsaturated hydrocarbon
group having 2 to 10 carbon atoms, X.sup.1 and X.sup.2 are each
independently an imino group (--NR.sup.1--), an ether group
(--O--), or a thioether group (--S--), and the R.sup.1 is a
hydrogen atom or a straight-chain or branched, saturated or
unsaturated hydrocarbon group having 2 to 10 carbon atoms.
[0008] The polynucleotide synthesis reagent of the present
invention includes a nucleotide derivative or a salt thereof
including the nucleoside derivative or a salt thereof of the
present invention.
[0009] The method for producing a polynucleotide of the present
invention includes the step of synthesizing a polynucleotide using
a nucleotide derivative or a salt thereof including the nucleoside
derivative or a salt thereof of the present invention.
[0010] The polynucleotide of the present invention includes, as a
building block, a nucleotide derivative or a salt thereof including
the nucleoside derivative or a salt thereof of the present
invention.
[0011] The method for producing a binding nucleic acid molecule of
the present invention includes the steps of: causing a candidate
polynucleotide and a target to come into contact with each other;
and selecting the candidate polynucleotide bound to the target as a
binding nucleic acid molecule that binds to the target, and the
candidate polynucleotide is the polynucleotide of the present
invention.
Advantageous Effects of Invention
[0012] The present invention can provide a novel nucleoside
derivative or a salt thereof.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1 is a graph showing the binding ability of an
.alpha.-amylase-binding nucleic acid molecule to .alpha.-amylase in
Example 2.
[0014] FIG. 2 is a graph showing the binding ability of the
secretory immunoglobulin A (sIgA)-binding nucleic acid molecule to
sIgA in Example 2.
[0015] FIG. 3 is a photograph showing the results of capillary
electrophoresis in Example 2.
[0016] FIG. 4 is a photograph showing the results of the pull-down
assay in Example 2.
[0017] FIGS. 5A to 5C are graphs showing the binding ability of the
respective types of .beta.-defensin (BDN)4A-binding nucleic acid
molecules to BDN4A in Example 3.
[0018] FIGS. 6A to 6C are graphs showing the binding ability of the
respective types of lysozyme-binding nucleic acid molecules to
lysozyme in Example 3.
[0019] FIG. 7 is a photograph showing the results of the pull-down
assay in Example 3.
[0020] FIGS. 8A and 8B are photographs showing the results of the
pull-down assay in Example 3.
[0021] FIGS. 9A and 9F are graphs showing the binding ability of
the respective types of .alpha.-amylase-binding nucleic acid
molecules to .alpha.-amylase in Example 4.
[0022] FIG. 10 is a photograph showing the results of capillary
electrophoresis in Example 4.
[0023] FIG. 11 is a photograph showing the results of the pull-down
assay in Example 4.
[0024] FIGS. 12A to 12D are graphs showing the binding ability of
the respective types of lactate dehydrogenase (LDH) 5-binding
nucleic acid molecules to LDHS in Example 5.
[0025] FIGS. 13A and 13B are graphs showing the binding ability of
the respective types of interleukin (IL) 6-binding nucleic acid
molecules to IL-6 in Example 5.
[0026] FIG. 14 is a graph showing the relative values of the
binding amounts of the respective types of LDHS-binding nucleic
acid molecules to LDHS in Example 5.
[0027] FIG. 15 is a graph showing the relative values of the
binding amounts of the respective types of IL-6 binding nucleic
acid molecules to IL-6 in Example 5.
[0028] FIG. 16 is a photograph showing the results of the pull-down
assay in Example 5.
DESCRIPTION OF EMBODIMENTS
[0029] (Nucleoside Derivative or Salt Thereof)
[0030] The nucleoside derivative or a salt thereof of the present
invention is represented by the following chemical formula (1), as
mentioned above.
##STR00003##
In the chemical formula (1), Su is an atomic group having a sugar
skeleton at a nucleoside residue or an atomic group having a sugar
phosphate skeleton at a nucleotide residue, and may or may not have
a protecting group, L.sup.1 and L.sup.2 are each independently a
straight-chain or branched, saturated or unsaturated hydrocarbon
group having 2 to 10 carbon atoms, X.sup.1 and X.sup.2 are each
independently an imino group (--NR.sup.1--), an ether group
(--O--), or a thioether group (--S--), and the R.sup.1 is a
hydrogen atom or a straight-chain or branched, saturated or
unsaturated hydrocarbon group having 2 to 10 carbon atoms.
[0031] The nucleoside derivative of the present invention has two
purine ring-like structures. The nucleoside derivative of the
present invention thus has, for example, a relatively larger number
of atoms capable of interacting within or between molecules than a
nucleoside derivative having one purine ring-like structure. The
binding nucleic acid molecule including the nucleoside derivative
of the present invention therefore has an improved binding ability
to a target, for example, compared to a nucleoside derivative
having one purine ring-like structure. Thus, with the nucleoside
derivative of the present invention, a binding nucleic acid
molecule that exhibits excellent binding ability to a target can be
produced, for example.
[0032] In the chemical formula (1), L.sup.1 and L.sup.2 are each
independently a straight-chain or branched, saturated or
unsaturated hydrocarbon group having 2 to 10 carbon atoms. The
lower limit of the number of carbon atoms of L.sup.1 is 2, the
upper limit of the same is 10, preferably 8 or 6, and the range of
the same is, for example, 2 to 8, 2 to 6. The number of carbon
atoms of L.sup.1 is preferably 2. The lower limit of the number of
carbon atoms of L.sup.2 is 2, the upper limit of the same is 10,
preferably 8 or 6, and the range of the same is, for example, 2 to
8, 2 to 6. The number of carbon atoms of L.sup.2 is preferably 2.
Specific examples of L.sup.1 and L.sup.2 include an ethylene group
(--CH.sub.2--CH.sub.2--), a vinylene group (--CH.dbd.CH--), a
propylene group (--CH.sub.2--CH.sub.2--CH.sub.2--), an isopropylene
group (--CH.sub.2--CH(CH.sub.3)-), a butylene group
(--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--), a methylbutylene
group (--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.2--), a
dimethylbutylene group
(--CH.sub.2--CH(CH.sub.3)--CH(CH.sub.3)--CH.sub.2--), an
ethylbutylene group
(--CH.sub.2--CH(C.sub.2H.sub.5)--CH.sub.2--CH.sub.2--), a pentylene
group (--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--), a
hexylene group
(--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--), a
heptylene group
(--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2---
), and an octylene group
(--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2--CH.sub.2---
CH.sub.2--). L.sup.1 is preferably a vinylene group
(--CH.dbd.CH--). L.sup.2 is preferably an ethylene group
(--CH.sub.2--CH.sub.2--). L.sup.1 and L.sup.2 may be the same
hydrocarbon group or different hydrocarbon groups. As a specific
example of the latter, L.sup.1 is preferably a vinylene group
(--CH.dbd.CH--), and L.sup.2 is preferably an ethylene group
(--CH.sub.2--CH.sub.2--).
[0033] In the chemical formula (1), X.sup.1 and X.sup.2 are each
independently an imino group (--NR.sup.1--), an ether group
(--O--), or a thioether group (--S--). In the imino group, the
R.sup.1 is a hydrogen atom or a straight-chain or branched,
saturated or unsaturated hydrocarbon group having 2 to 10 carbon
atoms and is preferably a hydrogen atom. The description of L.sup.1
and L.sup.2 can be incorporated in the description of the
straight-chain or branched, saturated or unsaturated hydrocarbon
group having 2 to 10 carbon atoms by reference. X.sup.1 is
preferably an imino group (--NR.sup.1--). X.sup.2 is preferably an
imino group (--NR.sup.1--). X.sup.1 and X.sup.2 may be the same
substituent or different substituents. As a specific example of the
former, X.sup.1 and X.sup.2 is preferably an imino group
(--NR.sup.1--) and more preferably an NH group.
[0034] In the chemical formula (1), the atomic group having a sugar
skeleton at a nucleoside residue is not particularly limited, and
examples thereof include atomic groups having sugar skeletons on
known natural or artificial nucleoside residues. Examples of the
atomic group having a sugar skeleton at a natural nucleoside
residue include an atomic group having a ribose skeleton at a
ribonucleoside residue and an atomic group having a deoxyribose
skeleton on a deoxyribonucleoside. The atomic group having a sugar
skeleton at an artificial nucleoside residue can be, for example,
an atomic group having a bicyclic sugar skeleton at an artificial
nucleoside residue, and specific examples thereof can be an atomic
group having a ribose skeleton where an oxygen atom at 2'-position
and a carbon atom at 4' position of ENA (2'-O,4'-C-Ethylene-bridged
Nucleic Acids) or LNA (Locked Nucleic Acid) is crosslinked. The
atomic group having a sugar phosphate skeleton at a nucleotide
residue is not particularly limited, and examples thereof include
atomic groups having sugar phosphate skeletons at known natural or
artificial nucleotide residues. Examples of the atomic group having
a sugar phosphate skeleton at a natural nucleotide residue include
an atomic group having a ribose phosphate skeleton at a
ribonucleotide residue and an atomic group having a deoxyribose
phosphate skeleton on a deoxyribonucleotide. The atomic group
having a sugar phosphate skeleton at an artificial nucleoside
residue can be, for example, an atomic group having a bicyclic
sugar phosphate skeleton at an artificial nucleoside residue, and
specific examples thereof can be an atomic group having a ribose
phosphate skeleton where an oxygen atom at 2'-position and a carbon
atom at 4' position of 2'-O,4'-C-Ethylene-bridged Nucleic Acids
(ENA) or Locked Nucleic Acid (LNA) is crosslinked.
[0035] In the chemical formula (1), an atomic group having a sugar
skeleton at a nucleoside residue or an atomic group having a sugar
phosphate skeleton at a nucleotide residue is represented by
preferably the following chemical formula (2).
##STR00004##
In the chemical formula (2), R.sup.2 is a hydrogen atom, a
protecting group, or a group represented by the following chemical
formula (3), R.sup.3 is a hydrogen atom, a protecting group, or a
phosphoramidite group, R.sup.4 is a hydrogen atom, a fluorine atom,
a hydroxyl group, an amino group, or a mercapto group, and
##STR00005##
in the chemical formula (3), Y is an oxygen atom or a sulfur atom,
Z is a hydroxyl group or an imidazole group, and m is an integer of
1 to 10.
[0036] In the chemical formula (2), R.sup.2 is a hydrogen atom, a
protecting group, or a group represented by the following chemical
formula (3). The protecting group is not particularly limited and
can be, for example, a protecting group of a known hydroxyl group
used in nucleic acid synthesis methods, and as a specific example,
the protecting group can be an DMTr group
(4,4'-dimethoxy(triphenylmethyl) group). When R.sup.2 is a group
represented by the chemical formula (3), the nucleoside derivative
of the present invention can also be referred to as a nucleotide
derivative, for example.
[0037] In the chemical formula (2), R.sup.3 is a hydrogen atom, a
protecting group, or a phosphoramidite group. The protecting group
is not particularly limited, and, the description of R.sup.2 can be
incorporated in the description of the protecting group by
reference, for example. The phosphoramidite group is represented by
the chemical formula (5). When R.sup.3 is a phosphoramidite group,
the nucleoside derivative of the present invention can also be
referred to as a phosphoramidite compound of the nucleoside
derivative, for example. When R.sup.2 is a group represented by the
chemical formula (3), and R.sup.3 is a phosphoramidite group, the
nucleoside derivative of the present invention can also be referred
to as, for example, a phosphoramidite compound of the nucleotide
derivative.
##STR00006##
[0038] In the chemical formula (2), R.sup.4 is a hydrogen atom, a
fluorine atom, a hydroxyl group, an amino group, or a mercapto
group and is preferably a hydrogen atom or a hydroxyl group. When
R.sup.4 is a hydrogen atom, the nucleoside derivative of the
present invention has a deoxyribose skeleton as a sugar skeleton
and can be used for, for example, synthesis of DNAs. When R.sup.4
is a hydroxyl group, the nucleoside derivative of the present
invention has a ribose skeleton as a sugar skeleton and can be used
for, for example, synthesis of RNAs.
[0039] In the chemical formula (3), Y is an oxygen atom or a sulfur
atom. When Y is an oxygen atom, polynucleotide including, as a
building block, the nucleoside derivative of the present invention
can also be referred to as polynucleotide having a phosphodiester
bond. When Y is a sulfur atom, polynucleotide including, as a
building block, the nucleoside derivative of the present invention
can also be referred to as polynucleotide having a phosphorothioate
bond.
[0040] In the chemical formula (3), Z is a hydroxyl group or an
imidazole group. In the imidazole group, imidazole is bound to a
phosphate atom via a nitrogen atom at the 1-position, for
example.
[0041] In the chemical formula (3), m is an integer of 1 to 10,
preferably 1 to 3, 1 to 2, or 1.
[0042] The nucleoside derivative of the present invention is
represented by preferably the following chemical formula (4), (6),
(7), or (8). The respective nucleoside derivatives represented by
the following chemical formulae (4), (6), (7), and (8) are also
referred to as MK4, MK1, MK2, and MK3.
##STR00007## ##STR00008##
[0043] The nucleoside derivative or a salt thereof of the present
invention may be a stereoisomer such as enantiomers, tautomers,
geometric isomers, conformers, and optical isomers thereof, and
salts thereof. Specifically, in the chemical formula (2) and
chemical formulae described below, the sugar skeleton is D body,
but the nucleoside derivative of the present invention is not
limited thereto, and the sugar skeleton may be L body.
[0044] The salt of the nucleoside derivative of the present
invention may be an acid addition salt or a base addition salt.
Further, acid which forms the acid addition salt may be an
inorganic acid or an organic acid, and base which forms the base
addition salt may be an inorganic base or an organic base. The
inorganic acid is not particularly limited, and examples thereof
include sulfuric acid, phosphoric acid, hydrofluoric acid,
hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorous
acid, hypochlorous acid, hypobromous acid, hypoiodous acid,
fluorous acid, chlorous acid, bromous acid, iodous acid, fluorine
acid, chloric acid, bromic acid, iodine acid, perfluoric acid,
perchloric acid, perbromic acid, and periodic acid. The organic
acid is not particularly limited, and examples thereof include
p-toluenesulfonic acid, methanesulfonic acid, oxalic acid,
p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric
acid, benzoic acid, and acetic acid. The inorganic base is not
particularly limited, and examples thereof include ammonium
hydroxides, alkali metal hydroxides, alkaline earth metal
hydroxides, carbonates, and bicarbonates. More specific examples
thereof include sodium hydroxide, potassium hydroxide, potassium
carbonate, sodium carbonate, sodium bicarbonate, potassium hydrogen
carbonate, calcium hydroxide, and calcium carbonate. The organic
base is not particularly limited, and examples thereof include
ethanolamine, triethylamine, and
tris(hydroxymethyl)aminomethane.
[0045] The method for producing the nucleoside derivative of the
present invention is not particularly limited, and the nucleoside
derivative of the present invention can be produced by combining
known synthesis methods. As a specific example, the nucleoside
derivative of the present invention can be synthesized by, for
example, an amidation reaction between a nucleoside derivative into
which an acrylic acid structure is introduced and an adenine in
which a substituent having an amino group at a terminal thereof is
substituted with a hydrogen atom of an amino group, as in the
synthetic method of the example described below.
[0046] (Polynucleotide Synthesis Reagent)
[0047] The polynucleotide synthesis reagent (hereinafter also
referred to as "synthesis reagent") of the present invention
contains a nucleotide derivative or a salt thereof including the
nucleoside derivative or a salt thereof of the present invention,
as mentioned above. The synthesis reagent of the present invention
is characterized by containing the nucleoside derivative of the
present invention, and other composition and conditions are not
particularly limited. The description of the nucleoside derivative
or a salt thereof of the present invention can be incorporated in
the description of the synthesis reagent of the present invention
by reference, for example. The synthesis reagent of the present
invention can be described with reference to the description of the
polynucleotide of the present invention, for example.
[0048] In the synthesis reagent of the present invention, the
nucleoside derivative preferably contains at least one of the
phosphoramidite compound or the nucleotide derivative, for
example.
[0049] The synthesis reagent of the present invention may further
contain another reagent for use in synthesis of polynucleotide, for
example.
[0050] (Method for Producing Polynucleotide)
[0051] The method for producing a polynucleotide of the present
invention includes, as mentioned above, the step of synthesizing a
polynucleotide using a nucleotide derivative or a salt thereof
including the nucleoside derivative or a salt thereof of the
present invention. The method for producing a polynucleotide of the
present invention is characterized by using a nucleotide derivative
or a salt thereof including the nucleoside derivative or a salt
thereof of the present invention in the synthesis step, and other
steps and conditions are not particularly limited. The descriptions
of the nucleoside derivative or a salt thereof and the synthesis
reagent of the present invention can be incorporated in the method
for producing a polynucleotide of the present invention by
reference, for example. By the method for producing a
polynucleotide of the present invention, the polynucleotide of the
present invention to be described below can be produced, for
example.
[0052] In the method for producing a polynucleotide of the present
invention, the synthesis reagent of the present invention may be
used as the nucleotide derivative or a salt thereof including the
nucleoside derivative or a salt thereof of the present
invention.
[0053] In the synthesis step, the method for synthesizing the
polynucleotide is not particularly limited, and the polynucleotide
can be synthesized by a known polynucleotide synthesis method.
[0054] When the phosphoramidite compound is used as the nucleotide
derivative or a salt thereof, the polynucleotide can be synthesized
by a phosphoramidite method in the synthesis step.
[0055] The method for producing the polynucleotide of the present
invention may further include a step of purifying the
polynucleotide obtained in the synthesis step, for example. The
purification method in the purification step is not particularly
limited, and the polynucleotide can be purified by a known
purification method such as column chromatography.
[0056] (Polynucleotide)
[0057] As mentioned above, the polynucleotide of the present
invention includes, as a building block, a nucleotide derivative or
a salt thereof including the nucleoside derivative or a salt
thereof of the present invention. The method for producing a
polynucleotide of the present invention is characterized by using a
nucleotide derivative or a salt thereof including the nucleoside
derivative or a salt thereof of the present invention in the
synthesis step, and other steps and conditions are not particularly
limited. The descriptions of the nucleoside derivative or a salt
thereof, the polynucleotide synthesis reagent, and the method for
producing a polynucleotide of the present invention can be
incorporated in the description of the polynucleotide of the
present invention by reference, for example. With the
polynucleotide of the present invention, a binding nucleic acid
molecule that binds to a target can be produced, for example, as
mentioned below. In the polynucleotide of the present invention,
the building block means, for example, a part of the
polynucleotide.
[0058] The polynucleotide of the present invention has a structure
represented by the following chemical formula (9), for example. The
description of each substituent can be incorporated in the
description of each substituent in the chemical formula (9) by
reference, for example.
##STR00009##
[0059] The polynucleotide of the present invention can be, for
example, a binding nucleic acid molecule that binds to a target.
The target is not particularly limited and can be any target, and
as a specific example, the target can be a biomolecule. Examples of
the biomolecule include secretory immunoglobulin A (sIgA), an
amylase (e.g., .alpha.-amylase), chromogranin A, .beta.-defensin
(Defensin) 2, .beta.-defensin 4A, kallikrein, C-reactive proteins
(CRPs), calprotectin, Statherins, cortisol, melatonin, lysozyme,
lactate dehydrogenase (LDH)5, and interleukin (IL)-6. The binding
nucleic acid molecule can be produced by the method for producing a
binding nucleic acid molecule of the present invention to be
described below.
[0060] The polynucleotide of the present invention may further
include, for example, other nucleotide in addition to the
nucleotide derivative. Examples of the nucleotide include
deoxyribonucleotide and ribonucleotide. Examples of the
polynucleotide of the present invention include DNA consisting of
deoxyribonucleotide only, DNA/RNA including deoxyribonucleotide and
ribonucleotide, and RNA consisting of ribonucleotide only. Other
nucleotide may be, for example, a modified nucleotide.
[0061] Examples of the modified nucleotide include modified
deoxyribonucleotide and modified ribonucleotide. The modified
nucleotide can be, for example, a nucleotide with a modified sugar.
Examples of the sugar include deoxyribose and ribose. The modified
site in the nucleotide is not particularly limited, and may be, for
example, the 2'-position or the 4'-position of the sugar. Examples
of the modification include methylation, fluorination, amination,
and thiation. The modified nucleotide can be, for example, a
modified nucleotide with a pyrimidine base (pyrimidine nucleus) as
a base or a modified nucleotide with a purine base (purine nucleus)
as a base and is preferably the former. Hereinafter, a nucleotide
with a pyrimidine base is referred to as pyrimidine nucleotide, the
pyrimidine nucleotide modified is referred to as modified
pyrimidine nucleotide, a nucleotide with a purine base is referred
to as purine nucleotide, and the purine nucleotide modified is
referred to as modified purine nucleotide. Examples of the
pyrimidine nucleotide include an uracil nucleotide with uracil,
cytosine nucleotide with cytosine, and thymine nucleotide with
thymine. When the base in the modified nucleotide is a pyrimidine
base, it is preferable that the 2'-position and/or the 4'-position
of the sugar is modified, for example. Specific examples of the
modified nucleotide include modified nucleotides with the
2'-position of the ribose being modified, such as a
2'-methylated-uracil nucleotide, 2'-methylated-cytosine nucleotide,
2'-fluorinated-uracil nucleotide, 2'-fluorinated-cytosine
nucleotide, 2'-aminated-uracil nucleotide, 2'-aminated-cytosine
nucleotide, 2'-thiated-uracil nucleotide, and 2'-thiated-cytosine
nucleotide.
[0062] The base in the other nucleotide may be, for example, a
natural base (non-artificial base) such as adenine (A), cytosine
(C), guanine (G), thymine (T), and uracil (U), or a non-natural
base (artificial base). Examples of the artificial base include
modified bases and altered bases. The artificial base preferably
has the same function as the natural base (A, C, G, T, or U).
Example of the artificial base having the same function as the
natural base include artificial bases capable of binding to
cytosine (C) instead of guanine (G), capable of binding to guanine
(G) instead of cytosine (C), capable of binding to thymine (T) or
uracil (U) instead of adenine (A), capable of binding to adenine
(A) instead of thymine (T), and capable of binding to adenine (A)
instead of uracil (U). The modified base is not particularly
limited, and may be, for example, a methylated base, a fluorinated
base, aminated base, and thiated base. Specific examples of the
modified base include 2'-methyluracil, 2'-methylcytosine,
2'-fluorouracil, 2'-fluorocytosine, 2'-aminouracil,
2'-aminocytosine, 2'-thiouracil, and 2'-thiocytosine. In the
present invention, for example, the bases represented by A, G, C,
T, and U include the meaning of, in addition to the natural bases,
the artificial bases having the same functions as the natural
bases.
[0063] The polynucleotide of the present invention may further
include, for example, an artificial nucleic acid monomer in
addition to the nucleotide derivative. Examples of the artificial
nucleic acid monomer include peptide nucleic acids (PNAs), LNAs,
and ENAs. The base in the monomer residue is the same as described
above, for example.
[0064] The length of the polynucleotide of the present invention is
not particularly limited, and the lower limit thereof is, for
example, 10-mer, 20-mer, or 25-mer, the upper limit thereof is, for
example, 150-mer, 100-mer, or 70-mer, and the range thereof is, for
example, 10- to 150-mer, 20- to 100-mer, or 25- to 70-mer.
[0065] The polynucleotide of the present invention may further
include an additional sequence, for example. Preferably, the
additional sequence is bound to at least one of the 5' end or the
3' end, more preferably to the 3' end of the polynucleotide, for
example. The additional sequence is not particularly limited, and
the length thereof is also not particularly limited.
[0066] The polynucleotide of the present invention may further
include a labeling substance, for example. Preferably, the labeling
substance is bound to at least one of the 5' end or the 3' end,
more preferably to the 5' end of the polynucleotide, for example.
The labeling substance is not particularly limited, and examples
thereof include fluorescent substances, dyes, isotopes, and
enzymes. Examples of the fluorescent substances include pyrenes,
TAMRA, fluorescein, Cy.RTM.3 dyes, Cy.RTM.5 dyes, FAM dyes,
rhodamine dyes, Texas Red dyes, fluorophores such as JOE, MAX, HEX,
and TYE, and examples of the dyes include Alexa dyes such as
Alexa.RTM.488 and Alexa.RTM.647.
[0067] The labeling substance may, for example, be linked directly
to the nucleic acid molecule or linked indirectly via the
additional sequence.
[0068] The polynucleotide of the present invention can be used in
the state where it is immobilized on a carrier, for example. It is
preferable to immobilize either the 5' end or the 3' end, more
preferably the 3' end of the polynucleotide of the present
invention, for example. When the polynucleotide of the present
invention is immobilized, the polynucleotide may be immobilized
either directly or indirectly on the carrier, for example. In the
latter case, it is preferable to immobilize the nucleic acid
molecule via the additional sequence, for example.
[0069] (Method for Producing Binding Nucleic Acid Molecule)
[0070] The method for producing a binding nucleic acid molecule of
the present invention includes, as mentioned above, the steps of:
causing a candidate polynucleotide and a target to come into
contact with each other; and selecting the candidate polynucleotide
bound to the target as a binding nucleic acid molecule that binds
to the target, and the candidate polynucleotide is the
polynucleotide of the present invention. The method for producing a
binding nucleic acid molecule of the present invention is
characterized in that the candidate polynucleotide is the
polynucleotide of the present invention, for example, and other
steps, conditions, etc. are not particularly limited. The
descriptions of the nucleoside derivative or a salt thereof, the
synthesis reagent, the method for producing polynucleotide, and the
polynucleotide can be incorporated in the method for producing a
binding nucleic acid of the present invention by reference, for
example. In the method for producing a binding nucleic acid
molecule of the present invention, the candidate polynucleotide
includes, as a building block, a nucleotide derivative or a salt
thereof including the nucleoside derivative or a salt thereof of
the present invention. Thus, for example, a binding nucleic acid
molecule that exhibits excellent binding ability to a target can be
produced by the method for producing a binding nucleic acid
molecule of the present invention.
[0071] As to the binding nucleic acid molecule of the present
invention, the contact step and the selection step can be performed
by the SELEX method, for example.
[0072] The number of candidate polynucleotides in the contact step
is not particularly limited, and the number of candidate
polynucleotides in the contact step is, for example, 4.sup.20 to
4.sup.120 types (about 10.sup.12 to 10.sup.72) and 4.sup.30 to
4.sup.60 types (about 10.sup.18 to 10.sup.36).
[0073] In the contact step, a candidate polynucleotide and a target
are caused to come into contact with each other. Then, by the
contact, the candidate polynucleotide and the target are reacted to
form a complex between the candidate polynucleotide and the target.
The target to be used in the contact step may be, for example, the
target itself or a decomposition product thereof. The conditions
under which the candidate polynucleotide and the target are bound
are not particularly limited, and for example, the binding can be
performed by incubating the both in a solvent for a certain period
of time. The solvent is not particularly limited, and for example,
a solvent in which the binding of the both is retained is
preferable, and specific examples thereof include various buffer
solutions.
[0074] Next, in the selecting step, a candidate polynucleotide
bound to the target is selected as a binding nucleic acid molecule
that binds to the target. Specifically, a candidate polynucleotide
that forms a complex with the target is collected as the binding
nucleic acid molecule. A mixture of the candidate polynucleotide
and the target after the contact step contains, in addition to the
complex, a candidate polynucleotide that is not involved in
formation of the complex, for example. Thus, it is preferable that
the complex and unreacted candidate polynucleotide are separated
from each other from the mixture, for example. The separation
method is not particularly limited and can be, for example, a
method utilizing a difference in adsorbability between the target
and the candidate polynucleotide or a difference in molecular
weight between the complex and the candidate polynucleotide.
[0075] In addition to this method, the separation method can be,
for example, a method using a target immobilized on a carrier in
formation of the complex. That is, the target is immobilized on a
carrier in advance to contact between the carrier and the candidate
polynucleotide, thereby forming a complex the immobilized target
and the candidate polynucleotide. An unreacted candidate
polynucleotide that does not bind to the immobilized target is then
removed, and the complex between the target and the candidate
polynucleotide is dissociated from the carrier. The method for
immobilizing the target on a carrier is not particularly limited
and can be carried out by a known method. The carrier is not
particularly limited, and, a known carrier can be used.
[0076] In the above-described manner, the binding nucleic acid
molecule that binds to a target can be produced.
[0077] The method for producing a binding nucleic acid molecule of
the present invention may further include, for example, the step of
determining a base sequence of the selected binding nucleic acid
molecule. The method for determining the base sequence is not
particularly limited, and the base sequence can be determined by a
known base sequence determination method.
[0078] In the method for producing a binding nucleic acid molecule
of the present invention, for example, one set of the contact step
and the selection step may be performed for two or more cycles in
total, and a specific example thereof is 3 to 15 cycles.
[0079] (.alpha.-amylase-Binding Nucleic Acid Molecule)
[0080] The .alpha.-amylase-binding nucleic acid molecule
(hereinafter also referred to as ".alpha.-amylase nucleic acid
molecule") of the present invention includes the following
polynucleotide (a):
(a) a polynucleotide (a1): (a1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 1 and 11 to 16.
[0081] The .alpha.-amylase nucleic acid molecule of the present
invention can bind to .alpha.-amylase, as mentioned above. The
.alpha.-amylase is not particularly limited, and the
.alpha.-amylase may be derived from a human or a non-human animal,
for example. Examples of the non-human animal include mice, rats,
monkeys, rabbits, dogs, cats, horses, cows, and pigs. Amino acid
sequence information on human .alpha.-amylase is registered under
Accession No. P04745 in UniProt (http://www.uniprot.org/), for
example.
[0082] In the present invention, the expression "binds to
.alpha.-amylase" (and grammatical variations thereof) is also
referred to as "has binding ability to .alpha.-amylase" or "has
binding activity to .alpha.-amylase", for example. The binding
between the nucleic acid molecule of the present invention and the
.alpha.-amylase can be determined by surface plasmon resonance
(SPR) analysis or the like, for example. The analysis can be
performed using ProteON (trade name, BioRad), for example. Since
the .alpha.-amylase nucleic acid molecule of the present invention
binds to .alpha.-amylase, it can be used for detection of the
.alpha.-amylase, for example.
[0083] As mentioned above, the .alpha.-amylase nucleic acid
molecule of the present invention comprises the following
polynucleotide (a):
(a) a polynucleotide (a1): (a1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 1 and 11 to 16.
TABLE-US-00001 .alpha.-amylase-binding nucleic acid molecule 1 (SEQ
ID NO: 1) 5'-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG
GGGGTCATTTGAAACTACAATGGGCGGGCTTATC-3' .alpha.-amylase-binding
nucleic acid molecule 2 (SEQ ID NO: 11)
5'-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG
GGGGTCATTTGAAACTA-3' .alpha.-amylase-binding nucleic acid molecule
3 (SEQ ID NO: 12) 5'-GCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATT
TGAAACTA-3' .alpha.-amylase-binding nucleic acid molecule 4 (SEQ ID
NO: 13) 55'-GGTTTGGACGCAATCTCCCTAATCAGACTATTATTTCAAGTAC
GTGGGGGTCTTGAAACTACAATGGGCGGGCTTATC-3' .alpha.-amylase-binding
nucleic acid molecule 5 (SEQ ID NO: 14)
5'-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG
GCGGCATTCAGAAACTACAATGGGCGGGCTTATC-3' .alpha.-amylase-binding
nucleic acid molecule 6 (SEQ ID NO: 15)
5'-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG GCGGCATTCAGAAACT-3'
.alpha.-amylase-binding nucleic acid molecule 7 (SEQ ID NO: 16)
5'-GCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTC AGAAACT-3''
[0084] The polynucleotide (a) above also includes, for example, the
meaning of the polynucleotide of (a2), (a3), or (a4) below:
(a2) a polynucleotide consisting of a base sequence obtained by
deletion, substitution, insertion, and/or addition of one or more
bases in any of the base sequences of the polynucleotide (a1) and
binds to the .alpha.-amylase; (a3) a polynucleotide consisting of a
base sequence having at least 80% sequence identity to any of the
base sequences of the polynucleotide (a1) and binds to the
.alpha.-amylase; and (a4) a polynucleotide consisting of a base
sequence complementary to a polynucleotide hybridizing to any of
the base sequences of the polynucleotide (a1) under stringent
conditions and binds to the .alpha.-amylase.
[0085] Regarding the polynucleotide (a2), the term "one or more" is
not limited as long as, for example, it is in the range where the
polynucleotide (a2) binds to .alpha.-amylase. The number of the
"one or more" bases is, for example, 1 to 15, 1 to 10, 1 to 7, 1 to
5, 1 to 3, 1 or 2, or 1. In the present invention, the numerical
range regarding the number of bases, sequences, or the like
discloses, for example, all the positive integers falling within
that range. That is, for example, the description "one to five
bases" discloses all of "one, two, three, four, and five bases"
(the same applies hereinafter).
[0086] Regarding the polynucleotide (a3), the "sequence identity"
is not limited as long as, for example, it is in the range where
the polynucleotide (a3) binds to .alpha.-amylase. The sequence
identity is, for example, at least 80%, at least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98%, or at least
99%. The sequence identity can be calculated with analysis software
such as BLAST or FASTA using default parameters, for example (the
same applies hereinafter).
[0087] Regarding the polynucleotide (a4), the "polynucleotide
hybridizing to" may be, for example, a polynucleotide that is
perfectly or partially complementary to the polynucleotide (a1) and
binds to the .alpha.-amylase. The hybridization can be detected by
various types of hybridization assay, for example. The
hybridization assay is not particularly limited, and for example, a
method described in "Molecular Cloning: A Laboratory Manual
2.sup.nd Ed." edited by Sambrook et al. (Cold Spring Harbor
Laboratory Press (1989)) or the like can be employed.
[0088] Regarding the polynucleotide (a4), the "stringent
conditions" may be any of low stringency conditions, medium
stringency conditions, and high stringency conditions, for example.
The "low stringency conditions" are, for example, conditions where
5.times.SSC, 5.times. Denhardt's solution, 0.5% SDS, and 50%
formamide are used at 32.degree. C. The "medium stringency
conditions" are, for example, conditions where 5.times.SSC,
5.times. Denhardt's solution, 0.5% SDS, and 50% formamide are used
at 42.degree. C. The "high stringency conditions" are, for example,
conditions where 5.times.SSC, 5 x Denhardt's solution, 0.5% SDS,
and 50% formamide, are used at 50.degree. C. Those skilled in the
art can set the degree of stringency by, for example, setting the
conditions such as the temperature, the salt concentration, the
concentration and length of a probe, the ionic strength, the time,
etc. as appropriate. As the "stringent conditions", it is also
possible to employ conditions described in the above-described
"Molecular Cloning: A Laboratory Manual 2.sup.nd Ed." edited by
Sambrook et al. (Cold Spring Harbor Laboratory Press (1989)) or the
like, for example.
[0089] In the .alpha.-amylase nucleic acid molecule of the present
invention, the building blocks of the polynucleotide are, for
example, nucleotide residues, examples of which include
deoxyribonucleotide residues and ribonucleotide residues. The
polynucleotide is, for example, a DNA consisting of
deoxyribonucleotide residues or a DNA including a
deoxyribonucleotide residue(s) and a ribonucleotide residue(s), and
the polynucleotide may further include a non-nucleotide residue(s),
as mentioned below. The .alpha.-amylase-binding nucleic acid
molecule of the present invention may be also referred to as
".alpha.-amylase aptamer" hereinafter, for example.
[0090] The .alpha.-amylase nucleic acid molecule of the present
invention may consist of any of the above-described
polynucleotides, or may include any of the above-described
polynucleotides, for example. In the latter case, the
.alpha.-amylase nucleic acid molecule of the present invention may
include, for example, two or more polynucleotides selected from the
above-described polynucleotides, as mentioned below. The two or
more polynucleotides may be the polynucleotides with the same
sequence or different sequences. Also, in the latter case, the
.alpha.-amylase nucleic acid molecule of the present invention may
further include a linker(s) and/or an additional sequence(s), for
example. The linker is a sequence present between polynucleotides,
for example. The additional sequence is a sequence added to an end,
for example.
[0091] When the .alpha.-amylase nucleic acid molecule of the
present invention includes, for example, a plurality of
polynucleotides selected from the above-described polynucleotides,
it is preferable that the plurality of polynucleotide sequences are
linked to each other to form a single-stranded polynucleotide. The
plurality of polynucleotide sequences may be linked to each other
directly, or may be linked to each other indirectly with a linker,
for example. It is preferable that the polynucleotide sequences are
linked to each other directly or indirectly at their ends. When the
.alpha.-amylase nucleic acid molecule of the present invention
includes the plurality of polynucleotide sequences, the number of
the sequences is not particularly limited, and is, for example, 2
or more, 2 to 20, 2 to 10, or 2 or 3.
[0092] The length of the linker is not particularly limited, and
is, for example, 1- to 200-mer, 1-to 20-mer, 3- to 12-mer, or 5- to
9-mer. The building blocks of the linker are, for example,
nucleotide residues, examples of which include deoxyribonucleotide
residues and ribonucleotide residues. The linker is not
particularly limited, and examples thereof include polynucleotides
such as a DNA consisting of deoxyribonucleotide residues and a DNA
including a ribonucleotide residue(s). Specific examples of the
linker include polydeoxythymine (poly[dT]), polydeoxyadenine
(poly[dA]), and poly(dA-dT) having a repetitive sequence composed
of A and T. Preferably, the linker is poly(dT) or poly(dA-dT).
[0093] In the .alpha.-amylase nucleic acid molecule of the present
invention, the polynucleotide is preferably a single-stranded
polynucleotide. It is preferable that the single-stranded
polynucleotide can form a stem structure and a loop structure by
self-annealing, for example. It is preferable that the
polynucleotide can form a stem-loop structure, an internal loop
structure, and/or a bulge structure, for example.
[0094] The .alpha.-amylase nucleic acid molecule of the present
invention may be a double strand, for example. When the
.alpha.-amylase nucleic acid molecule is a double strand, for
example, one of single-stranded polynucleotides includes the
polynucleotide (a), and the other single-stranded polynucleotide is
not limited. The other single-stranded polynucleotide may be, for
example, a polynucleotide including a base sequence complementary
to the polynucleotide (a). When the .alpha.-amylase nucleic acid
molecule of the present invention is a double strand, it is
preferable to dissociate the double strand to single-stranded
polynucleotides by denaturation or the like before use, for
example. Also, it is preferable that the dissociated
single-stranded polynucleotide including the polynucleotide (a) is
forming a stem structure and a loop structure as mentioned above,
for example.
[0095] In the present invention, the expression "can form a stem
structure and a loop structure" encompasses that, for example, a
stem structure and a loop structure are formed actually, and also,
even if a stem structure and a loop structure are not formed, they
can be formed depending on conditions. The expression "can form a
stem structure and a loop structure (and grammatical variations
thereof)" encompasses, for example, both the cases where the
formation thereof has been confirmed through an experiment and
where the formation thereof is predicted through simulation using a
computer or the like.
[0096] The building blocks of the .alpha.-amylase nucleic acid
molecule of the present invention are, for example, nucleotide
residues. Examples of the nucleotide residues include
deoxyribonucleotide residues and ribonucleotide residues. The
.alpha.-amylase nucleic acid molecule of the present invention may
be, for example, a DNA consisting of deoxyribonucleotide residues
only or a DNA including one or more ribonucleotide residues. In the
latter case, "one or more" is not particularly limited. For
example, the number of the ribonucleotide residues in the
polynucleotide is, for example, 1 to 91, 1 to 30, 1 to 15, 1 to 7,
1 to 3, or 1 or 2.
[0097] The polynucleotide may include, as a base in a nucleotide
residue, a natural base or a modified base. The natural base
(non-artificial base) is not particularly limited, and may be, for
example, a purine base with a purine skeleton or a pyrimidine base
with a pyrimidine skeleton. The purine base is not particularly
limited, and examples thereof include adenine (A) and guanine (G).
The pyrimidine base is not particularly limited, and examples
thereof include cytosine (C), thymine (T), and uracil (U). Among
them, cytosine (C) and thymine (T) are preferable.
[0098] When the polynucleotide includes the modified base(s), the
site and the number of the modified bases are not particularly
limited. When the polynucleotide (a) has the modified base(s), some
or all of the underlined adenines in the polynucleotide consisting
of any of base sequences of SEQ ID NOs: 1 and 11 to 16 are modified
bases, for example. When the underlined adenine is the modified
base, the modified base is a modified purine base, which is a
purine base modified with a modifying group.
[0099] The modified base is a base modified with a modifying group,
for example. The base to be modified with the modifying group (also
referred to simply as the "base to be modified" hereinafter) is the
natural base, for example. The natural base is not particularly
limited, and may be, for example, a purine base or a pyrimidine
base. The modified base is not particularly limited, and may be,
for example, a modified adenine, a modified guanine, a modified
cytosine, a modified thymine, or a modified uracil.
[0100] In the modified base, the base to be modified may be
modified with the modifying group either directly or indirectly,
for example. In the latter case, the base to be modified may be
modified with the modifying group via a linker, for example. The
linker is not particularly limited.
[0101] In the base to be modified, a site to be modified with the
modifying group is not particularly limited. When the base is a
purine base, the modified site in the purine base may be, for
example, the 7-position or the 8-position, preferably the
7-position of the purine skeleton. When the modified site in the
purine base is the 7-position of the purine skeleton, the nitrogen
atom at the 7-position is preferably substituted with a carbon
atom. When the base is a pyrimidine base, the modified site in the
pyrimidine base may be, for example, the 5-position or the
6-position, preferably the 5-position of the pyrimidine skeleton.
Thymine has a methyl group bound to carbon at the 5-position. Thus,
when the 5-position of the pyrimidine base is modified, for
example, the modifying group may be bound to the carbon at the
5-position either directly or indirectly, or the modifying group
may be bound to carbon in the methyl group bound to the carbon at
the 5-position either directly or indirectly. When the pyrimidine
skeleton has ".dbd.O" bound to carbon at the 4-position and a group
that is not "--CH.sub.3" or "--H" bound to carbon at the
5-position, the modified base can be referred to as a modified
uracil or a modified thymine.
[0102] When the modified base is a modified purine base, the
modifying group is preferably an adenine residue. That is, the
modified purine base is a base modified with an adenine residue,
for example. In the base to be modified, a site to be modified with
the adenine residue (binding site of the adenine residue to the
base to be modified) is not particularly limited, and can be, for
example, an amino group that binds to carbon at the 6-position of
the adenine residue. The base to be modified with the adenine
residue is not particularly limited, and is preferably purine base,
for example, and it is preferable that atom at the 7-position of
the purine base is modified with the adenine residue. When the
modified base is a modified thymine base, the modifying group is
preferably an adenine residue or a guanine base. That is, the
modified base is, for example, a base modified with an adenine
residue or a guanine residue. In the base to be modified, a site to
be modified with the adenine residue is not particularly limited,
and can be, for example, an amino group that binds to carbon at the
6-position of the adenine residue. In the base to be modified, a
site to be modified with the guanine residue is not particularly
limited, and can be, for example, an amino group that binds to
carbon at the 2-position of the guanine residue. The base to be
modified with the adenine residue or the guanine residue is not
particularly limited, and is preferably a thymine, for example, and
it is preferable that carbon in a methyl group bound to the carbon
at the 5-position of the thymine is modified with the adenine
residue or the guanine residue.
[0103] When the modifying group is the adenine residue or the
guanine residue, it is preferable that, for example, the base to be
modified is modified with the modifying group via the linker, as
shown below.
[nucleotide residue]-[linker]-[adenine residue] [nucleotide
residue]-[linker]-[guanine residue]
[0104] The linker is not particularly limited, and can be
represented by, for example, each formula present between the
nucleotide residue and the adenine residue/guanine residue, as
shown below. It is to be noted, however, that the linker is not
limited thereto. In each formula, the numerical value "n" in
(CH.sub.2).sub.n is 1 to 10, 2 to 10, or 2, for example.
[nucleotide
residue].dbd.C--C(.dbd.O)--NH--(CH.sub.2).sub.n-[adenine residue]
[nucleotide
residue].dbd.C--C(.dbd.O)--NH--(CH.sub.2).sub.n-[guanine residue]
[nucleotide
residue]C.dbd.C--C(.dbd.O)--NH--(CH.sub.2).sub.n-[adenine residue]
[nucleotide
residue].dbd.C--C(.dbd.O)--NH--CH.sub.2--CH.sub.2-[adenine residue]
[nucleotide
residue].dbd.C--C(.dbd.O)--NH--CH.sub.2--CH.sub.2-[guanine residue]
[nucleotide
residue]--C.dbd.C--C(.dbd.O)--NH--CH.sub.2--CH.sub.2-[adenine
residue]
[0105] In each formula, one ends of the linker [.dbd.C] and [--C]
form a double bond and a single bond with carbon of the base to be
modified in the nucleotide residue, respectively, for example, and
the other end of the linker [CH.sub.2--] is bound to amine (--NH)
in the guanine residue or the adenine residue, for example.
[0106] Specific examples of an adenosine nucleotide residue
modified with the adenine residue in the polynucleotide include a
residue represented by the following chemical formula (10) (also
referred to as "nucleotide residue of MK4" hereinafter). It is to
be noted, however, that the present invention is not limited
thereto.
##STR00010##
[0107] In the polynucleotide consisting of any of base sequences of
SEQ ID NOs: 1 and 11 to 16, it is more preferable that the
underlined adenine is a nucleotide residue of the MK4.
[0108] When the .alpha.-amylase nucleic acid molecule of the
present invention includes the nucleotide residues of the MK4, the
polynucleotide can be synthesized using, as a monomer molecule, a
nucleotide triphosphate represented by the following chemical
formula (4) (hereinafter also referred to as "MK4 monomer"
hereinafter), for example. In the synthesis of the polynucleotide,
for example, the monomer molecule binds to another nucleotide
triphosphate via a phosphodiester bond. A method for producing the
MK4 monomer is described below.
[0109] Other examples of the modifying group include a methyl
group, a fluoro group, an amino group, a thio group, a
benzylaminocarbonyl group, a tryptaminocarbonyl group, and an
isobutylaminocarbonyl group.
[0110] Specific examples of the modified adenine include
7'-deazaadenine. Specific examples of the modified guanine include
7'-deazaguanine. Specific examples of the modified cytosine include
5'-methylcytosine (5-Me-dC). Specific examples of the modified
thymine include 5'-benzylaminocarbonyl thymine,
5'-tryptaminocarbonyl thymine, and 5'-isobutylaminocarbonyl
thymine. Specific examples of the modified uracil include
5'-benzylaminocarbonyl uracil (BndU), 5'-tryptaminocarbonyl uracil
(TrpdU), and 5'-isobutylaminocarbonyl uracil. The modified uracils
given above as examples can be also referred to as modified
thymines.
[0111] The polynucleotide may include only one type or two or more
types of the modified bases, for example.
[0112] The .alpha.-amylase nucleic acid molecule of the present
invention may include a modified nucleotide, for example. The
modified nucleotide may be a nucleotide having the above-described
modified base, a nucleotide having a modified sugar obtained
through modification of a sugar residue, or a nucleotide having the
modified base and the modified sugar.
[0113] The sugar residue is not particularly limited, and may be a
deoxyribose residue or a ribose residue, for example. The modified
site in the sugar residue is not particularly limited, and may be,
for example, the 2'-position or the 4'-position of the sugar
residue. Either one or both of the 2'-position and the 4'-position
may be modified. Examples of a modifying group in the modified
sugar include a methyl group, a fluoro group, an amino group, a
thio group.
[0114] When the base in the modified nucleotide residue is a
pyrimidine base, it is preferable that the 2'-position and/or the
4'-position of the sugar residue is modified, for example. Specific
examples of the modified nucleotide residue include modified
nucleotide residues with the 2'-position of the deoxyribose residue
or ribose residue being modified, such as a 2'-methylated-uracil
nucleotide residue, 2'-methylated-cytosine nucleotide residue,
2'-fluorinated-uracil nucleotide residue, 2'-fluorinated-cytosine
nucleotide residue, 2'-aminated-uracil nucleotide residue,
2'-aminated-cytosine nucleotide residue, 2'-thiated-uracil
nucleotide residue, and 2'-thiated-cytosine nucleotide residue.
[0115] The number of the modified nucleotides is not particularly
limited. For example, the number of the modified nucleotides in the
polynucleotide is, for example, 1 to 100, 1 to 90, 1 to 80, or 1 to
70. Also, the number of the modified nucleotides in the full-length
nucleic acid molecule including the polynucleotide is not
particularly limited, and is, for example, 1 to 91, 1 to 78, or in
the numerical ranges given above as examples of the number of the
modified nucleotides in the polynucleotide.
[0116] The .alpha.-amylase nucleic acid molecule of the present
invention may include, for example, one or more artificial nucleic
acid monomer residues. The term "one or more" is not particularly
limited, and may be, for example, 1 to 100, 1 to 50, 1 to 30, or 1
to 10 in the polynucleotide, for example. Examples of the
artificial nucleic acid monomer residue include peptide nucleic
acids (PNAs), locked nucleic acids (LNAs), and
2'-O,4'-C-ethylenebridged nucleic acids (ENAs). The nucleic acid in
the monomer residue is the same as described above, for
example.
[0117] It is preferable that the .alpha.-amylase nucleic acid
molecule of the present invention is resistant to nuclease, for
example. In order to allow the .alpha.-amylase nucleic acid
molecule of the present invention to have nuclease resistance, it
is preferable that the nucleic acid molecule of the present
invention includes the modified nucleotide residue(s) and/or the
artificial nucleic acid monomer residue(s), for example. Also, in
order to allow the .alpha.-amylase nucleic acid molecule of the
present invention to have nuclease resistance, the nucleic acid
molecule of the present invention may have polyethylene glycol
(PEG) of several tens of kDa, deoxythymidine, or the like bound to,
e.g., the 5' end or the 3' end thereof.
[0118] The .alpha.-amylase nucleic acid molecule of the present
invention may further include an additional sequence, for example.
Preferably, the additional sequence is bound to at least one of the
5' end and the 3' end, more preferably to the 3' end of the nucleic
acid molecule, for example. The additional sequence is not
particularly limited. The length of the additional sequence is not
particularly limited, and is, for example, 1- to 200-mer, 1- to
50-mer, 1- to 25-mer, or 18-to 24-mer. The building blocks of the
additional sequence are, for example, nucleotide residues, examples
of which include deoxyribonucleotide residues and ribonucleotide
residues. The additional sequence is not particularly limited, and
examples thereof include polynucleotides such as a DNA consisting
of deoxyribonucleotide residues and a DNA including a
ribonucleotide residue(s). Specific examples of the additional
sequence include poly(dT) and poly(dA).
[0119] The .alpha.-amylase nucleic acid molecule of the present
invention can be used in the state where it is immobilized on a
carrier, for example. It is preferable to immobilize either the 5'
end or the 3' end, more preferably the 3' end of the
.alpha.-amylase nucleic acid molecule of the present invention, for
example. When the .alpha.-amylase nucleic acid molecule of the
present invention is immobilized, the .alpha.-amylase nucleic acid
molecule may be immobilized either directly or indirectly on the
carrier, for example. In the latter case, it is preferable to
immobilize the .alpha.-amylase nucleic acid molecule via the
additional sequence, for example.
[0120] The method for producing the .alpha.-amylase nucleic acid
molecule of the present invention is not particularly limited. For
example, the .alpha.-amylase nucleic acid molecule of the present
invention can be synthesized by known methods such as: nucleic acid
synthesis methods utilizing chemical synthesis; and genetic
engineering procedures.
[0121] The .alpha.-amylase nucleic acid molecule of the present
invention exhibits binding properties to the .alpha.-amylase, as
mentioned above. Thus, use of the .alpha.-amylase nucleic acid
molecule of the present invention is not particularly limited, as
long as it is the use utilizing the binding properties of the
.alpha.-amylase nucleic acid molecule to the .alpha.-amylase. The
.alpha.-amylase nucleic acid molecule of the present invention can
be used in various methods as an alternative to, e.g., an antibody
against the .alpha.-amylase.
[0122] (.alpha.-amylase Analysis Sensor)
[0123] The .alpha.-amylase analysis sensor of the present invention
is a sensor for analyzing .alpha.-amylase and includes the
.alpha.-amylase-binding nucleic acid molecule of the present
invention. It is only required that the .alpha.-amylase analysis
sensor of the present invention includes the
.alpha.-amylase-binding nucleic acid molecule of the present
invention, and other configurations, conditions, etc. are not
particularly limited. By using the .alpha.-amylase analysis sensor
of the present invention, the .alpha.-amylase can be detected by,
for example, causing the .alpha.-amylase nucleic acid molecule to
bind to the .alpha.-amylase. The description of the
.alpha.-amylase-binding nucleic acid molecule of the present
invention can be incorporated in the description of the
.alpha.-amylase analysis sensor of the present invention by
reference, for example.
[0124] The .alpha.-amylase analysis sensor of the present invention
may be configured so that, for example, it further includes a
carrier, and the .alpha.-amylase-binding nucleic acid molecule is
disposed on the carrier. Preferably, the .alpha.-amylase-binding
nucleic acid molecule is immobilized on the carrier. The
immobilization of the .alpha.-amylase-binding nucleic acid molecule
on the carrier is as described above, for example. The method for
using the .alpha.-amylase analysis sensor of the present invention
is not particularly limited, and the description of the
.alpha.-amylase nucleic acid molecule of the present invention and
the following description of the method for analyzing
.alpha.-amylase of the present invention can be incorporated in the
description of the .alpha.-amylase analysis sensor of the present
invention by reference.
[0125] (Method for Analyzing .alpha.-amylase)
[0126] The method for analyzing .alpha.-amylase of the present
invention includes the step of causing a specimen and a nucleic
acid molecule to come into contact with each other to detect
.alpha.-amylase in the specimen, the nucleic acid molecule is the
.alpha.-amylase-binding nucleic acid molecule of the present
invention, and in the detection step, the nucleic acid molecule is
caused to bind to the .alpha.-amylase in the specimen, and the
.alpha.-amylase in the specimen is detected by detecting the
binding. The method for analyzing .alpha.-amylase of the present
invention is characterized in that it uses the .alpha.-amylase
nucleic acid molecule of the present invention, and other steps,
conditions, etc. are not particularly limited. In the method for
analyzing .alpha.-amylase of the present invention, the
.alpha.-amylase analysis sensor of the present invention may be
used as the .alpha.-amylase nucleic acid molecule of the present
invention. The descriptions of the .alpha.-amylase-binding nucleic
acid molecule and the .alpha.-amylase analysis sensor of the
present invention can be incorporated in the description of the
method for analyzing .alpha.-amylase of the present invention by
reference, for example.
[0127] The nucleic acid molecule of the present invention
specifically binds to .alpha.-amylase. Thus, according to the
present invention, it is possible to specifically detect
.alpha.-amylase in a specimen by detecting the binding between the
.alpha.-amylase and the nucleic acid molecule, for example.
Specifically, since the present invention can analyze the presence
or absence or the amount of .alpha.-amylase in a specimen, for
example, it can be said that the present invention can also perform
qualitative or quantitative analysis of the .alpha.-amylase.
[0128] In the present invention, the specimen is not particularly
limited. Examples of the specimen include saliva, urine, plasma,
and serum.
[0129] The specimen may be a liquid specimen or a solid specimen,
for example. The specimen is preferably a liquid specimen from the
viewpoint of ease of handling because the liquid specimen can be
caused to come into contact with the nucleic acid molecule more
easily, for example. In the case of the solid specimen, a liquid
mixture, a liquid extract, a solution, or the like of the solid
specimen prepared using a solvent may be used, for example. The
solvent is not particularly limited, and may be water,
physiological saline, or a buffer solution, for example.
[0130] The above-described detection step includes, for example: a
contact step of causing the specimen and the nucleic acid molecule
to come into contact with each other to cause the nucleic acid
molecule to bind to the .alpha.-amylase in the specimen; and a
binding detection step of detecting the binding between the
.alpha.-amylase and the nucleic acid molecule. The detection step
may further include, for example, the step of analyzing the
presence or absence or the amount of the .alpha.-amylase in the
specimen on the basis of the result obtained in the binding
detection step.
[0131] In the contact step, the method for causing the specimen and
the nucleic acid molecule to come into contact with each other is
not particularly limited. The contact between the specimen and the
nucleic acid molecule preferably is achieved in a liquid, for
example. The liquid is not particularly limited, and may be, for
example, water, physiological saline, or a buffer solution.
[0132] In the contact step, the conditions under which the contact
between the specimen and the nucleic acid molecule is caused are
not particularly limited. The contact temperature is, for example,
4.degree. C. to 37.degree. C., or 18.degree. C. to 25.degree. C.,
and the contact time is, for example, 10 to 120 minutes or 30 to 60
minutes.
[0133] In the contact step, the nucleic acid molecule may be an
immobilized nucleic acid molecule immobilized on a carrier or an
unimmobilized nucleic acid molecule in a free state, for example.
In the latter case, the nucleic acid molecule is caused to come
into contact with the specimen in a container, for example. The
nucleic acid molecule is preferably the immobilized nucleic acid
molecule from the viewpoint of favorable handleability, for
example. The carrier is not particularly limited, and may be, for
example, a substrate, beads, or a container. The container may be a
microplate or a tube, for example. The immobilization of the
nucleic acid molecule is as described above, for example.
[0134] The binding detection step is the step of detecting the
binding between the .alpha.-amylase in the specimen and the nucleic
acid molecule, as mentioned above. By detecting the presence or
absence of the binding between the .alpha.-amylase and the nucleic
acid molecule, it is possible to analyze the presence or absence of
the .alpha.-amylase in the specimen (qualitative analysis), for
example. Also, by detecting the degree of the binding (the binding
amount) of the .alpha.-amylase to the nucleic acid molecule, it is
possible to analyze the amount of the .alpha.-amylase in the
specimen (quantitative analysis), for example.
[0135] In the case where the binding between the .alpha.-amylase
and the nucleic acid molecule cannot be detected, it can be
determined that no .alpha.-amylase is present in the specimen. In
the case where the binding is detected, it can be determined that
the .alpha.-amylase is present in the specimen.
[0136] The method for analyzing the binding between the
.alpha.-amylase and the nucleic acid molecule is not particularly
limited. A conventionally known method for detecting the binding
between substances may be employed as the method, for example, and
specific examples of the method include the above-described SPR.
Detection of the binding may be detection of a complex of the
.alpha.-amylase and the nucleic acid molecule, for example.
[0137] (.alpha.-amylase Detection Kit)
[0138] A .alpha.-amylase detection kit of the present invention
includes the .alpha.-amylase-binding nucleic acid molecule of the
present invention. It is only required that the .alpha.-amylase
detection kit of the present invention includes the
.alpha.-amylase-binding nucleic acid molecule of the present
invention, and other configurations, conditions, etc. are not
particularly limited. By using the .alpha.-amylase detection kit of
the present invention, it is possible to perform the detection and
the like of the .alpha.-amylase as mentioned above, for example.
The descriptions of the .alpha.-amylase-binding nucleic acid
molecule, the .alpha.-amylase analysis sensor, and the method for
analyzing .alpha.-amylase of the present invention can be
incorporated in the description of the .alpha.-amylase detection
kit by reference, for example.
[0139] The .alpha.-amylase detection kit of the present invention
may include the .alpha.-amylase analysis sensor of the present
invention as the .alpha.-amylase nucleic acid molecule of the
present invention, for example. The .alpha.-amylase detection kit
of the present invention may further include any component(s) in
addition to the .alpha.-amylase nucleic acid molecule of the
present invention, for example. Examples of the component include
the above-described carrier, a buffer solution, and instructions
for use.
[0140] (BDN4A-binding Nucleic Acid Molecule)
[0141] The .beta.-defensin (BDN)4A-binding nucleic acid molecule
(hereinafter also referred to as "BDN4A nucleic acid molecule") of
the present invention includes the following polynucleotide
(b):
(b) a polynucleotide (b1): (b1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 4 to 6.
[0142] The descriptions of the .alpha.-amylase-binding nucleic acid
molecule, the .alpha.-amylase analysis sensor, the method for
analyzing .alpha.-amylase, and the .alpha.-amylase detection kit
can be incorporated in the description of the BDN4A-binding nucleic
acid molecule of the present invention by reference, by, for
example, reading ".alpha.-amylase" as "BDN4A", reading "(a)" as
"(b)", reading "(a1)" as "(b1)", reading "(a2)" as "(b2)", reading
"(a3)" as "(b3)", reading "(a4)" as "(b4)", and reading "SEQ ID
NOs: 1 and 11 to 16" as "SEQ ID NOs: 4 to 6", unless otherwise
specifically stated. The same applies to the descriptions of the
BDN4A analysis sensor, the method for analyzing BDN4A, and the
BDN4A detection kit, to be described below.
[0143] The BDN4A nucleic acid molecule of the present invention can
bind to BDN4A, as mentioned above. The BDN4A is not particularly
limited, and the BDN4A may be derived from a human or a non-human
animal, for example. Examples of the non-human animal include mice,
rats, monkeys, rabbits, dogs, cats, horses, cows, and pigs. Amino
acid sequence information on human BDN4A is registered under
Accession No. 015263 in UniProt (http://www.uniprot.org/), for
example.
[0144] In the present invention, the expression "binds to BDN4A"
(and grammatical variations thereof) is also referred to as "has
binding ability to BDN4A" or "has binding activity to BDN4A", for
example. The binding between the BDN4A-binding nucleic acid
molecule of the present invention and the BDN4A can be determined
by surface plasmon resonance (SPR) analysis or the like, for
example. The analysis can be performed using ProteON (trade name,
BioRad), for example. Since the BDN4A nucleic acid molecule of the
present invention binds to BDN4A, it can be used for detection of
the BDN4A, for example.
[0145] As mentioned above, the BDN4A nucleic acid molecule of the
present invention includes the following polynucleotide (b):
(b) a polynucleotide (1): (b1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 4 to 6.
TABLE-US-00002 BDN4A-binding nucleic acid molecule 1 (SEQ ID NO: 4)
5'-GGTTACACGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCT
CTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3' BDN4A-binding nucleic acid
molecule 2 (SEQ ID NO: 5)
5'-CGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGA
GGAGTTCCCAGGCGAAGTTGTTATC-3' BDN4A-binding nucleic acid molecule 3
(SEQ ID NO: 6) 5'-GGTTACACGAGCCGCACATTTCACCGTGATAGTTCTCTGAGGAG
GACTTCTAGAGTTCCCAGGCGAAGTTGTTATC-3'
[0146] The polynucleotide (b) above also includes, for example, the
meaning of the polynucleotide of (b2), (b3), or (b4) below:
(b2) a polynucleotide consisting of a base sequence obtained by
deletion, substitution, insertion, and/or addition of one or more
bases in any of the base sequences of the polynucleotide (b1) and
binds to the BDN4A. (b3) a polynucleotide consisting of a base
sequence having at least 80% sequence identity to any of the base
sequences of the polynucleotide (b1) and binds to the BDN4A. (b4) a
polynucleotide consisting of a base sequence complementary to a
polynucleotide hybridizing to any of the base sequences of the
polynucleotide (b1) under stringent conditions and binds to the
BDN4A.
[0147] (BDN4A Analysis Sensor)
[0148] The .beta.-defensin (BDH)4A analysis sensor of the present
invention is a sensor for analyzing .beta.-defensin (BDN)4A and
includes the BDN4A-binding nucleic acid molecule of the present
invention. It is only required that the BDN4A analysis sensor of
the present invention includes the BDN4A-binding nucleic acid
molecule of the present invention, and other configurations,
conditions, etc. are not particularly limited. By using the BDN4A
analysis sensor of the present invention, the BDN4A can be detected
by, for example, causing the BDN4A nucleic acid molecule to bind to
the BDN4A. The description of the BDN4A-binding nucleic acid
molecule of the present invention can be incorporated in the
description of the BDN4A analysis sensor of the present invention
by reference, for example. The method for using the BDN4A analysis
sensor of the present invention is not particularly limited, and
the description of the BDN4A-binding nucleic acid molecule of the
present invention and the following description of the method for
analyzing BDN4A of the present invention can be incorporated in the
description of the BDN4A analysis sensor of the present invention
by reference.
[0149] (Method for Analyzing BDN4A)
[0150] The method for analyzing .beta.-defensin (BDH)4A of the
present invention includes the step of causing a specimen and a
nucleic acid molecule to come into contact with each other to
detect .beta.-defensin (BDN)4A in the specimen, the nucleic acid
molecule is the BDN4A-binding nucleic acid molecule of the present
invention, and in the detection step, the nucleic acid molecule is
caused to bind to the BDN4A in the specimen, and the BDN4A in the
specimen is detected by detecting the binding. The method for
analyzing BDN4A of the present invention is characterized in that
it uses the BDN4A-binding nucleic acid molecule of the present
invention, and other steps, conditions, etc. are not particularly
limited. In the method for analyzing BDN4A of the present
invention, the BDN4A analysis sensor of the present invention may
be used as the BDN4A nucleic acid molecule of the present
invention. The descriptions of the BDN4A-binding nucleic acid
molecule and the BDN4A analysis sensor of the present invention can
be incorporated in the description of the method for analyzing
BDN4A of the present invention by reference, for example.
[0151] (BDN4A Detection Kit)
[0152] The .beta.-defensin (BDN)4A detection kit of the present
invention includes the BDN4A-binding nucleic acid molecule of the
present invention. It is only required that the BDN4A detection kit
of the present invention includes the BDN4A-binding nucleic acid
molecule of the present invention, and other configurations,
conditions, etc. are not particularly limited. By using the BDN4A
detection kit of the present invention, it is possible to perform
the detection and the like of the BDN4A as mentioned above, for
example. The descriptions of the BDN4A-binding nucleic acid
molecule, the BDN4A analysis sensor, and the method for analyzing
BDN4 of the present invention can be incorporated in the
description of the BDN4A detection kit of the present invention by
reference.
[0153] (Lysozyme-binding Nucleic Acid Molecule)
[0154] The lysozyme binding nucleic acid molecule (hereinafter also
referred to as a "lysozyme nucleic acid molecule") of the present
invention includes the following polynucleotide (1):
(1) a polynucleotide (11): (11) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 7 to 9.
[0155] The descriptions of the .alpha.-amylase-binding nucleic acid
molecule, the .alpha.-amylase analysis sensor, the method for
analyzing .alpha.-amylase, and the .alpha.-amylase detection kit
can be incorporated in the description of the lysozyme-binding
nucleic acid molecule of the present invention by reference, by,
for example, reading ".alpha.-amylase" as "lysozyme", reading "(a)"
as "(1)", reading "(a1)" as "(11)", reading "(a2)" as "(12)",
reading "(a3)" as "(13)", reading "(a4)" as "(14)", and reading
"SEQ ID NOs: 1 and 11 to 16" as "SEQ ID NOs: 7 to 9", unless
otherwise specifically stated. The same applies to the descriptions
of the lysozyme analysis sensor, the method for analyzing lysozyme,
and the lysozyme detection kit, to be described below.
[0156] The lysozyme nucleic acid molecule of the present invention
can bind to lysozyme, as mentioned above. The lysozyme is not
particularly limited, and the lysozyme may be derived from a human
or a non-human animal, for example. Examples of the non-human
animal include mice, rats, monkeys, rabbits, dogs, cats, horses,
cows, and pigs. Amino acid sequence information on human lysozyme
is registered under Accession No. P61626 in UniProt
(http://www.uniprot.org/), for example.
[0157] In the present invention, the expression "binds to lysozyme"
(and grammatical variations thereof) is also referred to as "has
binding ability to lysozyme" or "has binding activity to lysozyme",
for example. The binding between the lysozyme nucleic acid molecule
of the present invention and the lysozyme can be determined by
surface plasmon resonance (SPR) analysis or the like, for example.
The analysis can be performed using ProteON (trade name, BioRad),
for example. Since the lysozyme nucleic acid molecule of the
present invention binds to lysozyme, it can be used for detection
of the lysozyme, for example.
[0158] As mentioned above, the lysozyme nucleic acid molecule of
the present invention includes the following polynucleotide
(l):
(l) a polynucleotide (l1): (l1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 7 to 9.
TABLE-US-00003 Lysozyme-binding nucleic acid molecule 1 (SEQ ID NO:
7) 5'-GGTTACACGAGCCGCACATTTCTAACGGGAACTTCAACCCATAC
AGTCTTTTGAGTTCCCAGGCGAAGTTGTTATC-3' Lysozyme-binding nucleic acid
molecule 2 (SEQ ID NO: 8)
5'-CGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTT TGAGTTCCC-3'
Lysozyme-binding nucleic acid molecule 3 (SEQ ID NO: 9)
5'-GGTTACACGAGCCGCACATTTCTTTACTCCGGAACCCATACAGT
CTTTTCCGGAGTTCCCAGGCGAAGTTGTTATC-3'
[0159] The polynucleotide (l) above also includes, for example, the
meaning of the polynucleotide of (l2), (l3), or (l4) below:
(l2) a polynucleotide consisting of a base sequence obtained by
deletion, substitution, insertion, and/or addition of one or more
bases in any of the base sequences of the polynucleotide (l1) and
binds to the lysozyme. (l3) a polynucleotide consisting of a base
sequence having at least 80% sequence identity to any of the base
sequences of the polynucleotide (l1) and binds to the lysozyme.
(l4) a polynucleotide consisting of a base sequence complementary
to a polynucleotide hybridizing to any of the base sequences of the
polynucleotide (l1) under stringent conditions and binds to the
lysozyme.
[0160] (Lysozyme Analysis Sensor)
[0161] The lysozyme analysis sensor of the present invention is a
sensor for analyzing lysozyme and includes the lysozyme-binding
nucleic acid molecule of the present invention. It is only required
that the lysozyme analysis sensor of the present invention includes
the lysozyme-binding nucleic acid molecule of the present
invention, and other configurations, conditions, etc. are not
particularly limited. By using the lysozyme analysis sensor of the
present invention, the lysozyme can be detected by, for example,
causing the lysozyme nucleic acid molecule to bind to the lysozyme.
The description of the lysozyme-binding nucleic acid molecule of
the present invention can be incorporated in the description of the
lysozyme analysis sensor of the present invention by reference, for
example. The method for using the lysozyme analysis sensor of the
present invention is not particularly limited, and the description
of the lysozyme-binding nucleic acid molecule of the present
invention and the following description of the method for analyzing
lysozyme of the present invention can be incorporated in the
description of the lysozyme analysis sensor of the present
invention by reference.
[0162] (Method for Analyzing Lysozyme)
[0163] The method for analyzing lysozyme of the present invention
includes the step of causing a specimen and a nucleic acid molecule
to come into contact with each other to detect lysozyme in the
specimen, the nucleic acid molecule is the lysozyme-binding nucleic
acid molecule of the present invention, and in the detection step,
the nucleic acid molecule is caused to bind to the lysozyme in the
specimen, and the lysozyme in the specimen is detected by detecting
the binding.
[0164] The method for analyzing lysozyme of the present invention
is characterized in that it uses the lysozyme-binding nucleic acid
molecule of the present invention, and other steps, conditions,
etc. are not particularly limited. In the method for analyzing
lysozyme of the present invention, the lysozyme analysis sensor of
the present invention may be used as the lysozyme nucleic acid
molecule of the present invention. The descriptions of the
lysozyme-binding nucleic acid molecule and the lysozyme analysis
sensor of the present invention can be incorporated in the
description of the method for analyzing lysozyme of the present
invention by reference, for example.
[0165] (Lysozyme Detection Kit)
[0166] The lysozyme detection kit of the present invention includes
the lysozyme-binding nucleic acid molecule of the present
invention. It is only required that the lysozyme detection kit of
the present invention includes the lysozyme-binding nucleic acid
molecule of the present invention, and other configurations,
conditions, etc. are not particularly limited. By using the
lysozyme detection kit of the present invention, it is possible to
perform the detection and the like of the lysozyme as mentioned
above, for example. The descriptions of the lysozyme-binding
nucleic acid molecule, the lysozyme analysis sensor, and the method
for analyzing lysozyme of the present invention can be incorporated
in the description of the lysozyme detection kit by reference, for
example.
[0167] (LDHS-binding Nucleic Acid Molecule)
[0168] The lactate dehydrogenase (LDH)5-binding nucleic acid
molecule (hereinafter also referred to as a "LDH5 nucleic acid
molecule") of the present invention includes the following
polynucleotide (d):
(d) a polynucleotide (d1): (d1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 17 to 20.
[0169] The descriptions of the .alpha.-amylase-binding nucleic acid
molecule, the .alpha.-amylase analysis sensor, the method for
analyzing .alpha.-amylase, and the .alpha.-amylase detection kit
can be incorporated in the description of the LDH5-binding nucleic
acid molecule of the present invention by reference, by, for
example, reading ".alpha.-amylase" as "LDH5", reading "(a)" as
"(d)", reading "(a1)" as "(d1)", reading "(a2)" as "(d2)", reading
"(a3)" as "(d3)", reading "(a4)" as "(d4)", and reading "SEQ ID
NOs: 1 and 11 to 16" as "SEQ ID NOs: 17 to 20", unless otherwise
specifically stated. The same applies to the descriptions of the
LDH5 analysis sensor, the method for analyzing LDH5, and the LDH5
detection kit, to be described below.
[0170] The LDH5 nucleic acid molecule of the present invention can
bind to LDH5 as mentioned above. The LDH5 is not particularly
limited, and the LDH5 may be derived from a human or a non-human
animal, for example. Examples of the non-human animal include mice,
rats, monkeys, rabbits, dogs, cats, horses, cows, and pigs. Amino
acid sequence information on human LDH5 is registered under
Accession No. P00338 in UniProt (http://www.uniprot.org/), for
example.
[0171] In the present invention, the expression "binds to LDH5"
(and grammatical variations thereof) is also referred to as "has
binding ability to LDH5" or "has binding activity to LDH5", for
example. The binding between the LDH5 nucleic acid molecule of the
present invention and the LDH5 can be determined by surface plasmon
resonance (SPR) analysis or the like, for example. The analysis can
be performed using ProteON (trade name, BioRad), for example. Since
the LDH5 nucleic acid molecule of the present invention binds to
LDH5, it can be used for detection of the LDH5, for example.
[0172] As mentioned above, the LDH5 nucleic acid molecule of the
present invention includes the following polynucleotide (d):
(d) a polynucleotide (d1): (d1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 17 to 20.
TABLE-US-00004 LDH5-binding nucleic acid molecule 1 (SEQ ID NO: 17)
5'-GGAATTGACACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGA
TATCAGGTCTCCTAAGGCTGGCTGGCTACTATAC-3' LDH5-binding nucleic acid
molecule 2 (SEQ ID NO: 18)
5'-GGAATTGACACCTCGCCGTTTATGAGAGGGAGATCATCTCTCTG
GCGGACACAACCTAAGGCTGGCTGGCTACTATAC-3' LDH5-binding nucleic acid
molecule 3 (SEQ ID NO: 19)
5'-ACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTC TCCTAAGGCTGGC-3'
LDH5-binding nucleic acid molecule 4 (SEQ ID NO: 20)
5'-TGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGG C-3'
[0173] The polynucleotide (d) above also includes, for example, the
meaning of the polynucleotide of (d2), (d3), or (d4) below:
(d2) a polynucleotide consisting of a base sequence obtained by
deletion, substitution, insertion, and/or addition of one or more
bases in any of the base sequences of the polynucleotide (d1) and
binds to the LDH5. (d3) a polynucleotide consisting of a base
sequence having at least 80% sequence identity to any of the base
sequences of the polynucleotide (d1) and binds to the LDH5. (d4) a
polynucleotide consisting of a base sequence complementary to a
polynucleotide hybridizing to any of the base sequences of the
polynucleotide (d1 ) under stringent conditions and binds to the
LDH5.
[0174] (LDH5 Analysis Sensor)
[0175] The LDH5 analysis sensor of the present invention is a
sensor for analyzing LDH5 and includes the LDH5-binding nucleic
acid molecule of the present invention. It is only required that
the LDH5 analysis sensor of the present invention includes the
LDH5-binding nucleic acid molecule of the present invention, and
other configurations, conditions, etc. are not particularly
limited. By using the LDH5 analysis sensor of the present
invention, the LDH5 can be detected by, for example, causing the
LDH5 nucleic acid molecule to bind to the LDH5. The description of
the LDH5-binding nucleic acid molecule of the present invention can
be incorporated in the description of the LDH5 analysis sensor of
the present invention by reference, for example. The method for
using the LDH5 analysis sensor of the present invention is not
particularly limited, and the description of the LDH5-binding
nucleic acid molecule of the present invention and the following
description of the method for analyzing LDH5 of the present
invention can be incorporated in the description of the LDH5
analysis sensor of the present invention by reference.
[0176] (Method for Analyzing LDH5)
[0177] The method for analyzing LDH5 of the present invention
includes the step of causing a specimen and a nucleic acid molecule
to come into contact with each other to detect LDH5 in the
specimen, the nucleic acid molecule is the LDH5-binding nucleic
acid molecule of the present invention, and in the detection step,
the nucleic acid molecule is caused to bind to the LDH5 in the
specimen, and the LDH5 in the specimen is detected by detecting the
binding. The method for analyzing LDH5 of the present invention is
characterized in that it uses the LDH5-binding nucleic acid
molecule of the present invention, and other steps, conditions,
etc. are not particularly limited. In the method for analyzing LDH5
of the present invention, the LDH5 analysis sensor of the present
invention may be used as the LDH5 nucleic acid molecule of the
present invention. The descriptions of the LDH5-binding nucleic
acid molecule and the LDH5 analysis sensor of the present invention
can be incorporated in the description of the method for analyzing
LDH5 of the present invention by reference, for example.
[0178] (LDH5 Detection Kit)
[0179] The LDH5 detection kit of the present invention includes the
LDH5-binding nucleic acid molecule of the present invention. It is
only required that the LDH5 detection kit of the present invention
includes the LDH5-binding nucleic acid molecule of the present
invention, and other configurations, conditions, etc. are not
particularly limited. By using the LDH5 detection kit of the
present invention, it is possible to perform the detection and the
like of the LDH5 as mentioned above, for example. The descriptions
of the LDH5-binding nucleic acid molecule, the LDH5 analysis
sensor, and the method for analyzing LDH5 of the present invention
can be incorporated in the description of the LDH5 detection kit of
the present invention by reference.
[0180] (IL-6-binding nucleic acid molecule)
[0181] The interleukin (IL)-6-binding nucleic acid molecule
(hereinafter also referred to as "IL-6 nucleic acid molecule") of
the present invention includes the following polynucleotide
(i):
(i) a polynucleotide (i1): (i1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 21 and 22.
[0182] The descriptions of the .alpha.-amylase-binding nucleic acid
molecule, the .alpha.-amylase analysis sensor, the method for
analyzing .alpha.-amylase, and the .alpha.-amylase detection kit
can be incorporated in the description of the IL-6-binding nucleic
acid molecule of the present invention by reference, by, for
example, reading ".alpha.-amylase" as "IL-6", reading "(a)" as
"(i)", reading "(a1)" as "(i1)", reading "(a2)" as "(i2)", reading
"(a3)" as "(i3)", reading "(a4)" as "(i4)", and reading "SEQ ID
NOs: 1 and 11 to 16" as "SEQ ID NOs: 21 and 22", unless otherwise
specifically stated. The same applies to the descriptions of the
IL-6 analysis sensor, the method for analyzing IL-6, and the IL-6
detection kit, to be described below.
[0183] The IL-6 nucleic acid molecule of the present invention can
bind to IL-6, as mentioned above. The IL-6 is not particularly
limited, and the IL-6 may be derived from a human or a non-human
animal, for example. Examples of the non-human animal include mice,
rats, monkeys, rabbits, dogs, cats, horses, cows, and pigs. Amino
acid sequence information on human IL-6 is registered under
Accession No. P05231 in UniProt (http://www.uniprot.org/), for
example.
[0184] In the present invention, the expression "binds to IL-6"
(and grammatical variations thereof) is also referred to as "has
binding ability to IL-6" or "has binding activity to IL-6", for
example. The binding between the IL-6 nucleic acid molecule of the
present invention and the IL-6 can be determined by surface plasmon
resonance (SPR) analysis or the like, for example. The analysis can
be performed using ProteON (trade name, BioRad), for example. Since
the IL-6 nucleic acid molecule of the present invention binds to
IL-6, it can be used for detection of the IL-6, for example.
[0185] As mentioned above, the IL-6 nucleic acid molecule of the
present invention includes the following polynucleotide (i):
(i) a polynucleotide (i1): (i1) a polynucleotide consisting of any
of base sequences of SEQ ID NOs: 21 and 22.
TABLE-US-00005 IL-6-binding nucleic acid molecule 1 (SEQ ID NO: 21)
5'-GGAATTGACACCTCGCCGTTTATGAGTTCAATGGTATTGTATCG
ACTCTTCTCGCCTAAGGCTGGCTGGCTACTATAC-3' IL-6-binding nucleic acid
molecule 2 (SEQ ID NO: 22)
5'-ACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCT C-3'
[0186] The polynucleotide (i) above also includes, for example, the
meaning of the polynucleotide of (i2), (i3), or (i4) below.
(i2) a polynucleotide consisting of a base sequence obtained by
deletion, substitution, insertion, and/or addition of one or more
bases in any of the base sequences of the polynucleotide (i1) and
binds to the IL-6. (i3) a polynucleotide comprising a base sequence
having 80% or more identity to the base sequence of any one of the
above (i1) and binds to IL-6. (i4) a polynucleotide consisting of a
base sequence complementary to a polynucleotide hybridizing to any
of the base sequences of the polynucleotide (i1) under stringent
conditions and binds to the IL-6.
[0187] (IL-6 Analysis Sensor)
[0188] The IL-6 analysis sensor of the present invention is a
sensor for analyzing IL-6 and includes the IL-6-binding nucleic
acid molecule of the present invention. It is only required that
the IL-6 analysis sensor of the present invention includes the
IL-6-binding nucleic acid molecule of the present invention, and
other configurations, conditions, etc. are not particularly
limited. By using the IL-6 analysis sensor of the present
invention, the IL-6 can be detected by, for example, causing the
IL-6 nucleic acid molecule to bind to the IL-6. The description of
the IL-6-binding nucleic acid molecule of the present invention can
be incorporated in the description of the IL-6 analysis sensor of
the present invention by reference, for example. The method for
using the IL-6 analysis sensor of the present invention is not
particularly limited, and the description of the IL-6-binding
nucleic acid molecule of the present invention and the following
description of the method for analyzing IL-6 of the present
invention can be incorporated in the description of the IL-6
analysis sensor of the present invention by reference.
[0189] (Method for Analyzing IL-6)
[0190] The method for analyzing IL-6 of the present invention
includes the step of causing a specimen and a nucleic acid molecule
to come into contact with each other to detect IL-6 in the
specimen, the nucleic acid molecule is the IL-6-binding nucleic
acid molecule of the present invention, and in the detection step,
the nucleic acid molecule is caused to bind to the IL-6 in the
specimen, and the IL-6 in the specimen is detected by detecting the
binding. The method for analyzing IL-6 of the present invention is
characterized in that it uses the IL-6-binding nucleic acid
molecule of the present invention, and other steps, conditions,
etc. are not particularly limited. In the method for analyzing IL-6
of the present invention, the IL-6 analysis sensor of the present
invention may be used as the IL-6 nucleic acid molecule of the
present invention. The descriptions of the IL-6-binding nucleic
acid molecule and the IL-6 analysis sensor of the present invention
can be incorporated in the description of the method for analyzing
IL-6 of the present invention by reference, for example.
[0191] (IL-6 Detection Kit)
[0192] The IL-6 detection kit of the present invention includes the
IL-6-binding nucleic acid molecule of the present invention. It is
only required that the IL-6 detection kit of the present invention
includes the IL-6-binding nucleic acid molecule of the present
invention, and other configurations, conditions, etc. are not
particularly limited. By using the IL-6 detection kit of the
present invention, it is possible to perform the detection and the
like of the IL-6 as mentioned above, for example. The descriptions
of the IL-6-binding nucleic acid molecule, the IL-6 analysis
sensor, and the method for analyzing IL-6 of the present invention
can be incorporated in the description of the IL-6 detection kit of
the present invention by reference.
EXAMPLES
[0193] The present invention is described more specifically below
with reference to examples. It is to be noted, however, that the
scope of the present invention is not limited by these examples.
Commercially available reagents in the examples were used in
accordance with their protocols, unless otherwise stated.
Example 1
[0194] MK1 to MK4 were prepared by the following synthesis
examples.
[0195] Electrospray ionization mass spectrometry (ESI-MS) was
performed using a mass spectrometer (API2000, vendor: Applied
Biosystems) in positive or negative ion mode. .sup.1H NMR spectra
were obtained using a nuclear magnetic resonance instrument
(JNM-ECS400, manufactured by JEOL). Chemical shifts are expressed
as relative .delta. (ppm) to the internal standard,
tetramethylsilane (Me.sub.4Si). Ion-exchange chromatography was
performed using a chromatographic system (ECONO system,
manufactured by Bio-Rad). In the ion-exchange chromatography, a
glass column (.phi.25.times.500 mm) packed with diethylaminoethyl
(DEAE) A-25-Sephadex (manufactured by Amershambiosciences) was
used.
Synthesis Example 1
Synthesis of MK1
##STR00011##
[0197] AZ6 (290 mg, 9.06.times.10.sup.-4 mol) was dried in vacuo
and dissolved in dry-DMF (N,N-dimethylformamide, 3 mL). To this
solution, HOBt.H.sub.2O (1-hydroxybenzotriazole monohydrate, 176
mg, 1.15.times.10.sup.-5 mol, 1.2 eq.), PyBOP.RTM.
(hexafluorophosphoric acid
(benzotriazole-1-yloxy)tripyrrolidinophosphonium, 579 mg,
1.15.times.10.sup.-5 mol, 1.2 eq.), and DIPEA
(N,N-diisopropylethylamine, 4.6 mL, 2.72.times.10.sup.-2 mol, 30
eq.) were added, and NK1 (493 mg, 9.48.times.10 mol, 1.1 eq.)
dissolved in dry-DMF (1 mL) was further added and stirred. After 40
minutes from the initiation of the stirring, the solvent was
distilled off under reduced pressure, and a residue was dissolved
in water, and a precipitate was collected by suction filtration.
The filtrate was roughly purified by reversed-phase column
chromatography, to give MK1.
Physical properties of MK1 are shown below. Yield amount: 261 mg,
Yield: 60% ESI-MS (positive ion mode) m/z, found =481.2, calculated
for [(M+H)+] =481.2 found =503.1, calculated for [(M+Na)+]
=503.2
.sup.1HNMR (400 MHz, DMSO-d6) .delta.8.22 (1H, m), 8.11 (1H, s),
8.10 (1H, s), 7.87 (1H, s), 7.63 (1H, d), 6.52 (1H, q), 6.35 (1H,
d), 5.27 (1H, s), 3.82 (1H, m), 2.18 (1H, m)
Synthesis Example 2
Synthesis of MK2
##STR00012##
[0199] MK1 (108 mg, 2.25.times.10.sup.4 mol) was dried in vacuo,
and the atmosphere was replaced with Ar (Argon). Subsequently,
azeotropy between the MK1 and dry-DMF was caused twice (the first
time: 40 mL, the second time: 4 mL), and azeotropy between the MK1
and dry-MeCN (acetonitrile) was caused three times (the first time:
9 mL, the second time: 5 mL, the third time: 5 mL). The resultant
azeotrope was suspended in dry-Trimethyl phosphate (6 mL), and
thereafter, dry-Tributhyl amine (130 .mu.L, 5.44.times.10.sup.4
mol, 2.5 eq.) was added thereto. Then, phosphoryl chloride (42
.mu.L, 4.50.times.10.sup.4 mol, 2 eq.) was added and stirred under
ice cooling.
[0200] After 40 minutes from the initiation of the stirring,
dry-Tributhyl amine (250 .mu.L, 1.05 .times.10.sup.-3 mol, 5 eq.)
and Phosphoryl chloride (84 .mu.L, 4.50.times.10.sup.4 mol, 4 eq.)
were again added and stirred under ice cooling for 1 hour. After
the stirring, a cooled 1 mol/L TEAB (Triethylammonium bicarbonate)
buffer (5 mL) was added, stirred for 5 minutes, and quenched. Then,
the solvent was distilled off under reduced pressure,
crystallization was performed in Ether, and suction filtration was
performed to obtain a yellow solid. The yellow solid was dissolved
in water, purified by anion-exchange column chromatography, and
freeze-dried, to give MK2. Physical properties of MK2 are shown
below.
Yield: 30.0 .mu.mol yield: 13.4% ESI-MS (negative ion mode) m/z,
found =559.1, calculated for [(M-H)-]=559.2
Synthesis Example 3
Synthesis of MK3
##STR00013##
[0202] MK2 (30.03 .sub.Ilmol) was dried in vacuo, and azeotropy
between the MK2 and dry-Pyridine (10 mL) was performed three times,
and the azeotrope was dried in vacuo overnight. After the drying,
the atmosphere was replaced with Ar, and the MK2 was dissolved in
dry-DMF (2 mL) and dry-TEA (triethylamine, 28 .mu.L,
1.98.times.10.sup.4 mol, 6.6 eq.). Further, Imidazole (16 mg,
14.02.times.10.sup.4 mol, 4 eq.), 2,2'-Dithiodipyridine (17 mg,
7.72.times.10 mol, 1.6 eq.), and Triphenylphosphine (20 mg,
7.63.times.10.sup.4 mol, 1.6 eq.) were added and stirred at room
temperature. After 6.5 hours from the initiation of the stirring,
the resultant reaction solution was added to a solution of Sodium
perchlorate (39 mg, 3.19.times.10.sup.4 mol, 10 eq.) in dry-Acetone
(18 mL), dry Ether (27 mL), and dry-TEA (2 mL), and allowed to
stand at 4.degree. C. for 30 minutes. The precipitate was decanted
5 times with dry-Ether (12 mL) and was thereafter dried in vacuo,
to give MK3 as a crude.
Theoretical yield amount: 30.03 .mu.mol
Synthesis Example 4
Synthesis of MK4
##STR00014##
[0204] MK3 (30.03 .mu.mol) was dried in vacuo, the atmosphere was
replaced with Ar, and then, azeotropy between the MK3 and
dry-Pyridine (5 mL) was caused twice, and the azeotrope was then
suspended in dry-DMF (1 mL). Further, dry-n-Tributylamine (30
.mu.L, 1.25.times.10.sup.4 mol, 4 eq.) and 0.5 mol/L
n-Tributylamine pyrophosphate in DMF (310 .mu.L,
1.53.times.10.sup.4 mol, 5 eq.) were added to the suspension and
then stirred at room temperature. After 6.5 hours from the
initiation of the stirring, a 1 mol/L TEAB buffer (5 mL) was added
and stirred for 30 minutes, and then the solvent was distilled off
under reduced pressure. Water was added, an aqueous layer was
separated with Ether twice, purified by anion-exchange column
chromatography, and freeze-dried, to give MK4. Physical properties
of MK4 are shown below.
Yield: 3.33 .mu.mol, Yield: 11.1% ESI-MS (negative ion mode) m/z,
found =719.0, calculated for [(M-H)-]=719.1
Example 2
[0205] The present example examined whether binding nucleic acid
molecules that bind to sIgA and binding nucleic acid molecules that
bind to .alpha.-amylase can be obtained using MK4.
[0206] (1) Binding Nucleic Acid Molecule
[0207] Binding nucleic acid molecules that bind to a target were
obtained by the SELEX method, except that candidate polynucleotides
prepared by using, in addition to deoxyribonucleotides containing
thymine, guanine, and cytosine, respectively (dTTP, and dGTP, and
dCTP, respectively), MK4 as deoxyribonucleotide were used.
Specifically, the binding nucleic acid molecules were obtained in
the following manner. sIgA (manufactured by MP Biomedicals,
LLC-Cappel Products) or human salivary amylase (manufactured by Lee
BioSolutions, Inc.) as the target was bound to beads (Dynabeads
MyOne Carboxylic Acid, manufactured by Invitrogen) according to the
protocols attached to the products. After binding the target, the
beads were washed with a selection buffer (SB Buffer: 40 mmol/L
HEPES, 125 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L MgCl.sub.2, 0.01%
Tween.RTM. 20, pH 7.5), whereby target beads were prepared. dsDNAs
with MK4 inserted therein were prepared using complementary strands
with their 5' ends modified with biotin (forward (Fw) primer
region-N30 (30 bases)-reverse (Rv) primer region), forward primers
and DNA polymerase (KOD Dash, manufactured by Toyobo Co., Ltd.),
and dTTP, dGTP, dCTP and MK4. Subsequently, the dsDNAs were bound
to the beads (Dynabeads MyOne Carboxylic Acid), and then, ss
(single strand) DNAs were eluted with a 0.02 mol/L NaOH aqueous
solution. Furthermore, the NaOH aqueous solution was neutralized
with a 0.08 mol/L hydrochloric acid aqueous solution. Thus, an
ssDNA library was prepared. 20 pmol of the library was mixed with
250 .sub.1.tg of the target beads at 25.degree. C. for 15 minutes.
Thereafter, the beads were washed with the SB buffer. Then, the
ssDNAs bound to the beads were eluted with a 7 mol/L urea aqueous
solution. The eluted ssDNAs were amplified by PCR using the forward
primers and the biotin-modified reverse primers. In the PCR, dTTP,
adenine-containing deoxyribonucleotide (dATP), dGTP, and dCTP were
used as deoxyribonucleotides. The obtained dsDNAs were bound to
magnetic beads (Dynabeads MyOne SA Cl magnetic beads, manufactured
by Invitrogen). Thereafter, forward strands were eluted with a 0.02
mol/L NaOH aqueous solution and removed. After removing the forward
strands, the magnetic beads were washed with the SB buffer. Using
the magnetic beads with the complementary strands immobilized
thereon, forward primers and DNA polymerase (KOD Dash, manufactured
by Toyobo Co., Ltd.), dTTP, dGTP, dCTP, and MK4, dsDNAs with MK4
inserted therein were prepared in the above-described manner. Next,
an ssDNA library was prepared by eluting forward strands with a
0.02 mol/L NaOH aqueous solution, and this library was used in a
subsequent round. Nucleic acid molecules that bind to sIgA or
.alpha.-amylase were selected by performing eight rounds of the
same process. Thereafter, PCR was performed using forward primers
and reverse primers without biotin modification. The obtained
nucleic acid molecules were subjected to sequencing using a
sequencer (GS junior sequencer, manufactured by Roche).
[0208] As a result, a binding nucleic acid molecule consisting of
the base sequence of SEQ ID NO: 1 below was obtained as the binding
nucleic acid molecule that binds to .alpha.-amylase, and a binding
nucleic acid molecule consisting of the base sequence of SEQ ID NO:
2 below was obtained as the binding nucleic acid molecule that
binds to sIgA. In the base sequences of SEQ ID NOs: 1 and 2, the
underlined bases A are MK4.
TABLE-US-00006 .alpha.-amylase-binding nucleic acid molecule 1 (SEQ
ID NO: 1) 5'-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG
GGGGTCATTTGAAACTACAATGGGCGGGCTTATC-3' sIgA-binding nucleic acid
molecule (SEQ ID NO: 2)
5'-GGTTTGGACGCAATCTCCCTAATCAAGCCACGGAGAGTCCGAGG
TGACCATTAAGCAGGAAACTACAATGGGCGGGCTTA-3'
[0209] (2) Examination of Binding by Surface Plasmon Resonance
(SPR)
[0210] The binding between the .alpha.-amylase-binding nucleic acid
molecule 1 and .alpha.-amylase and the binding between the
sIgA-binding nucleic acid molecule and sIgA were measured under the
following SPR conditions. The .alpha.-amylase-binding nucleic acid
molecule 1 and sIgA-binding nucleic acid molecule adapted so that a
20-mer poly(dT) was added to the 3' ends were each used as the
following ligand 2. Further, as control 1 and control 2,
examination of the binding was performed in the same manner except
that, in control 1, bovine serum albumin (BSA) was used as the
following analyte, and in control 2, chromogranin A (CgA,
manufactured by Creative BioMart) was used as the following analyte
in a system for examining the binding of the
.alpha.-amylase-binding nucleic acid molecules 1 and the
.alpha.-amylase was used as the following analyte in a system for
examining the binding of the sIgA-binding nucleic acid
molecules.
[0211] (SPR Measurement Conditions)
Measurement device: ProteOn.TM. XPR36 (manufactured by BioRad)
Measurement chip: ProteOn.TM. NLC Sensor Chip (manufactured by
BioRad) Ligand 1: poly(dA) (20-mer) with the 5' end thereof being
modified with biotin: 5 .mu.mol/L Buffer: 40 mmol/L HEPES, 125
mmol/L NaCl, 1 mmol/L MgCl.sub.2, 5 mmol/L KCl, 0.01%
Tween.RTM. 20, pH 7.4
[0212] Ligand 2: buffer containing a binding nucleic acid molecule
with poly(T) (20-mer) added to the 3' end at 200 nmol/L Ligand flow
rate: 25 .mu.L/min, 80 sec Analyte: buffer containing a target at
400 nmol/L Analyte flow rate: 50 .mu.L/min
Contact Time: 120 sec
Dissociation: 300 sec
[0213] sIgA: IgA (Secretory), Human (manufactured by MP
Biomedicals, LLC-Cappel Products, Catalogue number: #55905) [0214]
Amylase: .alpha.-amylase (manufactured by Lee Biosolutions,
Catalogue number: #120-10) [0215] CgA: recombinant full length
Human Chromogranin A (manufactured by Creative BioMart, Catalog
number: #CHGA 26904TH) [0216] BSA: Bovine Serum Albumin
(manufactured by SIGMA, Catalogue number: #A7906)
[0217] The results of measuring the binding between the
.alpha.-amylase-binding nucleic acid molecule 1 and the
.alpha.-amylase are shown in FIG. 1, and the results of measuring
the binding between the sIgA-binding nucleic acid molecule and sIgA
are shown in FIG. 2. FIG. 1 is a graph showing the binding ability
of the .alpha.-amylase-binding nucleic acid molecule 1 to the
.alpha.-amylase. In FIG. 1, the horizontal axis indicates the time
elapsed after the injection of the ligand, and the vertical axis
indicates the relative value (RU) of the binding force. As can be
seen in FIG. 1, the .alpha.-amylase-binding nucleic acid molecule 1
bound to the .alpha.-amylase, whereas they did not bind to CgA or
BSA.
[0218] Next, FIG. 2 is a graph showing the binding ability of the
sIgA-binding nucleic acid molecule to sIgA. In FIG. 2, the
horizontal axis indicates the time elapsed after the injection of
the ligand, and the vertical axis indicates the relative value (RU)
of the binding force. As can be seen in FIG. 2, the sIgA-binding
nucleic acid molecules bind sIgA, whereas they do not bind to the
.alpha.-amylase or BSA.
[0219] From these results, it was found that a binding nucleic acid
molecule that binds to .alpha.-amylase and a binding nucleic acid
molecule that binds to sIgA can be obtained using MK4, which is the
nucleoside derivative of the present invention.
[0220] (3) Examination of Binding Force
[0221] The relative value (RU) of the binding force was measured in
the same manner as in the above item (2), except that the
.alpha.-amylase-binding nucleic acid molecule 1 having a 20-mer
poly(T) added to its 3' end was used as the ligand 2 and that the
concentration of the .alpha.-amylase as the analyte was set to 5,
10, 20, 40, or 80 nmol/L. Also, the relative value (RU) of the
binding force was measured in the same manner as in the above item
(2), except that the sIgA-binding nucleic acid molecule having a
20-mer poly(T) added to its 3' end was used as the ligand 2 and
that the concentration of sIgA as the analyte was 12.5, 25, 50,
100, or 200 nmol/L. Then, based on the relative values (RU) of the
binding force measured in the above, the dissociation constant
between the .alpha.-amylase-binding nucleic acid molecule 1 and the
.alpha.-amylase and the dissociation constant between the
sIgA-binding nucleic acid molecule and sIgA were calculated. As a
result, the dissociation constant between the
.alpha.-amylase-binding nucleic acid molecules 1 and the
.alpha.-amylase was 8.14 nM, and the dissociation constant between
the sIgA-binding nucleic acid molecules and sIgA was 7.63 nM. These
results demonstrate that both the binding nucleic acid molecules
have excellent binding ability to the targets.
[0222] (4) Examination of Binding by Capillary Electrophoresis
[0223] Binding between the .alpha.-amylase-binding nucleic acid
molecule 1 and .alpha.-amylase was measured by capillary
electrophoresis performed under the following conditions. The
.alpha.-amylase-binding nucleic acid molecule adapted so that the
5' end thereof was labeled with a 20-mer TYE.TM. 665 was used as
the following clone. As a control, the measurement was performed in
the same manner except that .alpha.-amylase was not added as the
target.
[0224] (Conditions of Capillary Electrophoresis)
Measurement device: Cosmo-i SV1210 (Hitachi High-Technologies
Corporation) Measurement chip: i-chip 12 (Hitachi Chemical Company,
Ltd.) Electrophoresis gel: 0.6% (Hydroxypropyl)methyl cellulose,
viscosity 2. 600-5, 600 (manufactured by SIGMA, Catalogue number:
#H7509) Gel dissolving buffer: 40 mmol/L HEPES (pH 7.5), 5 mmol/L
KCl, 1 mmol/L MgCl.sub.2 Clone: solution containing 200 nmol/L
amylase-binding nucleic acid molecule with its 5' end labeled with
TYETM 665, 40 mmol/L HEPES (pH 7.5), 125 mmol/L NaCl, 5 mmol/L KCl,
and 1 mmol/L MgCl.sub.2 Target: solution containing 4 .mu.mol/L
amylase (a-Amylase-High Purity, Human, manufactured by Lee
BioSolutions, Inc., Catalogue number: #120-10), 40 mmol/L HEPES (pH
7.5), 125 mmol/L NaCl, 5 mmol/L KCl, and 1 mmol/L MgCl.sub.2
Folding: 95.degree. C., after 5 min, on ice 5 min Mixing: after
addition of target, room temperature (around 25.degree. C.), 30
min, 1000 rpm Injection voltage: 600 V Injection time: 120 sec
Separation voltage: 350 V
Separation Time: 260 sec
[0225] The results obtained are shown in FIG. 3. FIG. 3 is a
photograph showing the results of capillary electrophoresis. In
FIG. 3, the electrophoresis time is shown on the left side of the
photograph, and the respective lanes show, from the left, the
result obtained regarding the control (without .alpha.-amylase) and
the result obtained when the .alpha.-amylase was used. As can be
seen in FIG. 3, the electrophoresis time in the presence of the
.alpha.-amylase was longer than that in the control without
.alpha.-amylase. From these results, it was found that the
.alpha.-amylase-binding nucleic acid molecule 1 binds to
.alpha.-amylase.
[0226] (5) Examination of Binding by Pull-Down Assay
[0227] Beads 1 carrying .alpha.-amylase-binding nucleic acid
molecule bound thereto (hereinafter, also referred to as "bound
beads Al") were prepared by bringing an .alpha.-amylase-binding
nucleic acid molecule 1 with its 5' end modified with biotin into
contact with streptavidin-modified beads (Dynabeads MyOne SA C1
magnetic beads, manufactured by Invitrogen). Next, the bound beads
A1 were mixed with a saliva-containing SB buffer (saliva sample),
and the resultant mixture was shaken at 1000 rpm for 60 minutes at
room temperature (around 25.degree. C.).
[0228] After the shaking, the bound beads A1 were washed three
times with the SB buffer. Then, the bound beads A1 were treated at
95.degree. C. for 10 minutes in the presence of the SDS buffer,
whereby the .alpha.-amylase bound to the bound beads A1 was eluted.
The composition of the SDS buffer was as follows: 62.5 mmol/L Tris,
2% SDS, 5% sucrose, 0.002% Bromophenol blue, and 1%
2-mercaptoethanol.
[0229] The thus-obtained eluate was loaded onto a gel (PAGEL C5OL,
manufactured by ATTO), and electrophoresis was performed in the
presence of an electrophoresis buffer. The composition of the
electrophoresis buffer was as follows: 25 mmol/L Tris, 192 mmol/L
glycine, and 0.1% SDS. Next, the gel after the electrophoresis was
stained with a staining agent (Gel Code, manufactured by Thermo
SCIENTIFIC, Catalogue number: #24594), and imaged with ChemiDoc
(manufactured by BioRad). Further, as control 1 and control 2,
imaging was performed in the same manner except that, in control 1,
nucleic acid molecules that do not bind to .alpha.-amylase with
their 5' ends modified with biotin were used (control nucleic acid
molecules 1) and, in control 2, only .alpha.-amylase was used.
TABLE-US-00007 Control nucleic acid molecule 1 (SEQ ID NO: 3)
5'-GGATACCTTAACGCCGCCTATTG-3'
[0230] The results obtained are shown in FIG. 4. FIG. 4 is a
photograph showing the results of the pull-down assay. In FIG. 4,
the numerical values on the left side of the photograph indicate
molecular weights, and the respective lanes show, from the left,
the results obtained regarding the molecular weight marker (M), the
saliva sample (1), control 1 (C1), and control 2 (AMY). As can be
seen in FIG. 4, in the lane showing the result of control 1, no
band was observed at the same position (about 50 kDa) as in the
lane showing the result of control 2, whereas, in the lane showing
the result regarding the saliva sample, a band was observed at the
same electrophoretic mobility position as in the lane showing the
result of control 2. In other words, the binding of the
.alpha.-amylase-binding nucleic acid molecules 1 to .alpha.-amylase
was observed. From these results, it was found that the
.alpha.-amylase-binding nucleic acid molecule 1 binds to
.alpha.-amylase.
Example 3
[0231] The present example examined whether a binding nucleic acid
molecule that binds to human .beta.-defensin 4A and a binding
nucleic acid molecule that binds to human lysozyme can be obtained
using MK4.
[0232] (1) Binding Nucleic Acid Molecule
[0233] The binding nucleic acid molecule that binds to human
.beta.-defensin 4A and the binding nucleic acid molecule that binds
to human lysozyme were obtained in the same manner as in the above
item (1) in Example 2, except that, instead of sIgA as the target,
human .beta.-defensin 4A (manufactured by Novoprotein Scientific
Inc., Catalog number: #C127) or human lysozyme (manufactured by
Novoprotein Scientific Inc., Catalog number: #P61626) was used.
[0234] As a result, as the binding nucleic acid molecule that binds
to .beta.-defensin (BDN)4A, the binding nucleic acid molecules
consisting of the base sequences of SEQ ID NOs: 4 to 6 shown below
were obtained, and as the binding nucleic acid molecule that binds
to lysozyme, binding nucleic acid molecules consisting of the base
sequences of SEQ ID NOs: 7 to 9 shown below were obtained. In the
base sequences of SEQ ID NOs: 4 to 9 below, each underlined A is
MK4.
TABLE-US-00008 BDN4A-binding nucleic acid molecule 1 (SEQ ID NO: 4)
5'-GGTTACACGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCT
CTGAGGAGGAGTTCCCAGGCGAAGTTGTTATC-3' BDN4A-binding nucleic acid
molecule 2 (SEQ ID NO: 5)
5'-CGAGCCGCACATTTCTATTTTTACGGGGTATAGTTCTCTGAGGA
GGAGTTCCCAGGCGAAGTTGTTATC-3' BDN4A-binding nucleic acid molecule 3
(SEQ ID NO: 6) 5'-GGTTACACGAGCCGCACATTTCACCGTGATAGTTCTCTGAGGAG
GACTTCTAGAGTTCCCAGGCGAAGTTGTTATC-3' Lysozyme-binding nucleic acid
molecule 1 (SEQ ID NO: 7)
5'-GGTTACACGAGCCGCACATTTCTAACGGGAACTTCAACCCATAC
AGTCTTTTGAGTTCCCAGGCGAAGTTGTTATC-3' Lysozyme-binding nucleic acid
molecule 2 (SEQ ID NO: 8)
5'-CGAGCCGCACATTTCTAACGGGAACTTCAACCCATACAGTCTTT TGAGTTCCC-3'
Lysozyme-binding nucleic acid molecule 3 (SEQ ID NO: 9)
5'-GGTTACACGAGCCGCACATTTCTTTACTCCGGAACCCATACAGT
CTTTTCCGGAGTTCCCAGGCGAAGTTGTTATC-3'
[0235] (2) Examination of Binding by SPR
[0236] The binding between each type of the BDN4A-binding nucleic
acid molecules and BDN4A and the binding between each type of the
lysozyme-binding nucleic acid molecules and lysozyme were measured
in the same manner as in the above item (2) in Example 2, except
that the BDN4A-binding nucleic acid molecules or lysozyme-binding
nucleic acid molecules having a 20-mer poly(dT) added to their 3'
ends were used as the ligand 2 and each of the following proteins
was used as the analyte. As controls, the binding was examined in
the same manner except that .alpha.-amylase and sIgA were used as
the analytes. [0237] .beta.-defensin 4A: P-Defensin 4A, Human
(manufactured by Novoprotein Scientific Inc., Catalog number:
#C127) [0238] Human lysozyme: Recombinant Human Lysozyme C
(manufactured by Novoprotein Scientific Inc., Catalog number:
#P61626)
[0239] The results of measuring the binding between the respective
types of the BDN4A-binding nucleic acid molecules and BDN4A are
shown in FIGS. 5A to 5C. The results of measuring the binding
between the respective types of the lysozyme-binding nucleic acid
molecules and the lysozyme are shown in FIGS. 6A to 6C.
[0240] FIGS. 5A to 5C are graphs showing the binding ability of the
respective types of the BDN4A-binding nucleic acid molecules to
BDN4A. FIGS. 5A to 5C show the results obtained regarding the
BDN4A-binding nucleic acid molecules 1 to 3, respectively. In each
of FIGS. 5A to 5C, the horizontal axis indicates the time elapsed
after the injection of the ligand, and the vertical axis indicates
the relative value of the binding force (RU). As can be seen in
FIGS. 5A to 5C, the respective types of the BDN4A-binding nucleic
acid molecules did not bind to the amylase or sIgA and bound to
BDN4A.
[0241] Next, FIGS. 6A to 6C are graphs showing the binding ability
of the respective types of the lysozyme-binding nucleic acid
molecules to the lysozyme. FIGS. 6A to 6C show the results obtained
regarding the lysozyme-binding nucleic acid molecules 1 to 3,
respectively. In each of FIGS. 6A to 6C, the horizontal axis
indicates the time elapsed after the injection of the ligand, and
the vertical axis indicates the relative value (RU) of the binding
force. As can be seen in FIGS. 6A to 6C, the lysozyme-binding
nucleic acid molecules did not bind to the amylase or sIgA and
bound to the lysozyme.
[0242] From these results, it was found that a binding nucleic acid
molecule that binds to BDN4A and a binding nucleic acid molecule
that binds to lysozyme can be obtained using MK4, which is the
nucleoside derivative of the present invention.
[0243] (3) Examination of Binding Force
[0244] The relative value (RU) of the binding force was measured in
the same manner as in the above item (2) in Example 3, except that
the BDN4A-binding nucleic acid molecules having a 20-mer poly(T)
added to their 3' ends were used as the ligand 2 and that the
concentration of BDN4A as the analyte was set to 25, 50, 100, 200,
or 400 nmol/L. Also, the relative value (RU) of the binding force
was measured in the same manner as in the item (2) in Example 3,
except that each type of the lysozyme-binding nucleic acid
molecules having a 20-mer poly(T) added to their 3' ends were used
as the ligand 2 and that the concentration of the lysozyme as the
analyte was set to 12.5, 25, 50, 100, or 200 nmol/L. Then, on the
basis of the relative values (RU) of the binding force measured in
the above, the dissociation constant between each type of the
BDN4A-binding nucleic acid molecules and BDN4A and the dissociation
constant between each type of the lysozyme-binding nucleic acid
molecules and the lysozyme were calculated. The results obtained
are shown in Table 1 below.
TABLE-US-00009 TABLE 1 Dissociation Nucleic acid molecule name
constant (nM) BDN4A-binding nucleic acid molecule 1 6.52
BDN4A-binding nucleic acid molecule 2 8.07 BDN4A-binding nucleic
acid molecule 3 48.2 Lysozyme-binding nucleic acid molecule 1 1.18
Lysozyme-binding nucleic acid molecule 2 1.03 Lysozyme-binding
nucleic acid molecule 3 1.83
[0245] As can be seen in Table 1 above, it was found that these
binding nucleic acid molecules all have excellent binding ability
to the targets.
[0246] (4) Examination of Binding by Pull-Down Assay
[0247] Beads carrying BDN4A-binding nucleic acid molecules bound
thereto (also referred to as "bound beads C" hereinafter) and beads
carrying lysozyme-binding nucleic acid molecules bound thereto
(also referred to as "bound beads D" hereinafter) were prepared by
bringing BDN 4A-binding nucleic acid molecules 1 and
lysozyme-binding nucleic acid molecules 1 with their 5' ends
modified with biotin into contact with the above-described
streptavidin-modified beads, respectively. Next, SDS-PAGE was
performed, and gel was imaged in the same manner as in the item (5)
in Example 2, except that the bound beads C and D were each mixed
with a SB buffer containing 90 (v/v)% saliva (saliva sample) or a
SB buffer containing BDN4A or lysozyme (target sample). Further, as
control 1 and control 2, imaging was performed in the same manner
except that, in control 1, the following control nucleic acid
molecules 2 with their 5' ends modified with biotin were used and,
in control 2, only BDN4A or only lysozyme was used.
TABLE-US-00010 Control nucleic acid molecule 2 (SEQ ID NO: 10)
5'-GGTAACCGCCCTGTCTTGATAAC-3'
[0248] Next, the results obtained when the bound beads C were used
are shown in FIG. 7. FIG. 7 is a photograph showing the results of
the pull-down assay using the bound beads C. In FIG. 7, the
numerical values on the left side of the photograph indicate
molecular weights, and the respective lanes are, from the left,
lane M (marker), lane 4 (target sample), lane C1 (control 1), lane
hBDN (control 2), lane M (marker), lane 8 (saliva sample), lane C2
(control 1), and lane hBDN (control 2). As can be seen in FIG. 7,
in the lane showing the result of control 1, no band was observed
at the same position (about 10 kDa) as in the lane showing the
result of control 2, whereas, in the lanes showing the results
obtained when the target sample and the saliva sample were used,
bands were observed at the same electrophoretic mobility position
as in the lane showing the result of control 2, as indicated with
the arrows in FIG. 7. In other words, binding of the BDN4A-binding
nucleic acid molecules 1 to BDN4A was observed. From these results,
it was found that the BDN4A-binding nucleic acid molecules bind to
BDN4A.
[0249] Next, the results obtained when the bound beads D were used
are shown in FIGS. 8A and 8B. FIGS. 8A and 8B are photographs
showing the results of the pull-down assay. FIG. 8A shows the
result obtained when the target sample was used and FIG. 8B shows
the result obtained when the saliva sample was used. In FIG. 8A,
the numerical values on the left side of the photograph indicate
molecular weights, and the respective lanes are, from the left,
lane M (marker), lane 2 (target sample), lane C1 (control 1), lane
hLys (control 2), and lane M (marker). In FIG. 8B, the respective
lanes are, from the left, lane M (marker), lane 6 (saliva sample),
lane C1 (control 1), lane hLys (control 2), and lane M (marker). As
can be seen in FIG. 8, in the lane showing the result of control 1,
no band was observed at the same position (about 15 kDa) as in the
lane showing the result of control 2, whereas, in the lanes showing
the results obtained when the target sample and the saliva sample
were used, bands were observed at the same electrophoretic mobility
position as in the lane showing the result of control 2, as
indicated with the arrows in FIG. 8. In other words, binding of the
lysozyme-binding nucleic acid molecules 1 to the lysozyme was
observed. From these results, it was found that the
lysozyme-binding nucleic acid molecules bind to lysozyme.
Example 4
[0250] The present example examined whether binding nucleic acid
molecules that bind to human .alpha.-amylase can be obtained using
MK4.
[0251] (1) Binding Nucleic Acid Molecule
[0252] Binding nucleic acid molecules that bind to human
.alpha.-amylase were obtained in the same manner as in the item (1)
in Example 2, except that, instead of sIgA as the target, the
above-described human .alpha.-amylase was used.
[0253] As a result, as the binding nucleic acid molecules that bind
to .alpha.-amylase, binding nucleic acid molecules consisting of
the base sequences of SEQ ID NOs: 11 to 16 shown below were
obtained. In the base sequences of SEQ ID NOs: 11 to 16 below, each
underlined A is MK4.
TABLE-US-00011 .alpha.-amylase-binding nucleic acid molecule 2 (SEQ
ID NO: 11) 5'-GGTTTGGACGCAATCTCCCTAATCTAGTGACGAAAATGTACGAG
GGGGTCATTTGAAACTA-3' .alpha.-amylase-binding nucleic acid molecule
3 (SEQ ID NO: 12) 5'-GCAATCTCCCTAATCTAGTGACGAAAATGTACGAGGGGGTCATT
TGAAACTA-3' .alpha.-amylase-binding nucleic acid molecule 4 (SEQ ID
NO: 13) 5'-GGTTTGGACGCAATCTCCCTAATCAGACTATTATTTCAAGTACG
TGGGGGTCTTGAAACTACAATGGGCGGGCTTATC-3' .alpha.-amylase-binding
nucleic acid molecule 5 (SEQ ID NO: 14)
5'-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG
GCGGCATTCAGAAACTACAATGGGCGGGCTTATC-3' .alpha.-amylase-binding
nucleic acid molecule 6 (SEQ ID NO: 15)
5'-GGTTTGGACGCAATCTCCCTAATCTAAAGTTTCTAAACGATGTG GCGGCATTCAGAAACT-3'
.alpha.-amylase-binding nucleic acid molecule 7 (SEQ ID NO: 16)
5'-GCAATCTCCCTAATCTAAAGTTTCTAAACGATGTGGCGGCATTC AGAAACT-3'
[0254] (2) Examination of Binding by SPR
[0255] The binding between each type of the .alpha.-amylase-binding
nucleic acid molecules and the .alpha.-amylase was measured in the
same manner as for measuring the binding of the
.alpha.-amylase-binding nucleic acid molecules 1 in the above item
(2) in Example 2. As controls, the binding was examined in the same
manner except that CgA and BSA were used as the analytes. The
results obtained are shown in FIGS. 9A to 9F.
[0256] FIGS. 9A to 9F are graphs showing the binding ability of the
respective types of the .alpha.-amylase-binding nucleic acid
molecules to the .alpha.-amylase. In FIGS. 9A to 9F show the
results obtained regarding the .alpha.-amylase-binding nucleic acid
molecules 2 to 7, respectively. In each of FIGS. 9A to 9F, the
horizontal axis indicates the time elapsed after the injection of
the ligand, and the vertical axis indicates the relative value (RU)
of the binding force. As can be seen in FIGS. 9A to 9F, the
respective types of the .alpha.-amylase-binding nucleic acid
molecules did not bind to CgA or BSA and bound to the
.alpha.-amylase.
[0257] (3) Examination of Binding Force
[0258] The relative value (RU) of the binding force was measured in
the same manner as in the item (2) in Example 4, except that each
type of the .alpha.-amylase-binding nucleic acid molecules having a
20-mer poly(T) added to their 3' ends were used as the ligand 2,
and the concentration of the .alpha.-amylase as the analyte was set
to 5, 10, 20, 40, or 80 nmol/L. Then, on the basis of the relative
values (RU) of the binding force measured in the above, the
dissociation constant between each type of the
.alpha.-amylase-binding nucleic acid molecules and the
.alpha.-amylase was calculated. As a result, the dissociation
constants between the .alpha.-amylase-binding nucleic acid
molecules 2 to 7 and the .alpha.-amylase were 6.91, 7.75, 5.18,
13.2, 11.5, and 11.1 nM, respectively. These results demonstrate
that all of the binding nucleic acid molecules have excellent
binding ability to the targets.
[0259] (4) Examination of Binding by Capillary Electrophoresis
[0260] The binding was measured in the same manner as in the item
(4) in Example 2, except that, in addition to the
.alpha.-amylase-binding nucleic acid molecules 1, the
.alpha.-amylase-binding nucleic acid molecules 5 were used. As a
control, the measurement was performed in the same manner, except
that the .alpha.-amylase was not added as the target.
[0261] The results obtained are shown in FIG. 10. FIG. 10 is a
photograph showing the results of capillary electrophoresis. In
FIG. 10, the electrophoresis time is shown on the left side of the
photograph, and the respective lanes show, from the left, the
results obtained regarding the .alpha.-amylase-binding nucleic acid
molecules 1 in the control (without .alpha.-amylase) and in the
presence of the .alpha.-amylase, and the results obtained regarding
the .alpha.-amylase-binding nucleic acid molecules 5 in the control
(without .alpha.-amylase) and in the presence of the
.alpha.-amylase. As can be seen in FIG. 10, regarding the
respective types of the .alpha.-amylase-binding nucleic acid
molecules, the electrophoresis time in the presence of the
.alpha.-amylase was longer than that in the control without
.alpha.-amylase. From these results, it was found that the
.alpha.-amylase-binding nucleic acid molecules 1 and 5 bind to
.alpha.-amylase.
[0262] (5) Examination of Binding by Pull-Down Assay
[0263] Beads 5 carrying .alpha.-amylase-binding nucleic acid
molecules bound thereto (also referred to as "bound beads A5"
hereinafter) were prepared by bringing the .alpha.-amylase-binding
nucleic acid molecules 5 with their 5' ends modified with biotin
into contact with the above-described streptavidin-modified beads.
Then, imaging was performed in the same manner as in the item (5)
in Example 2, except that the bound beads A5 were used in addition
to the bound beads A1 and that a target sample containing
.alpha.-amylase was used as the sample. Further, as control 1 and
control 2, imaging was performed in the same manner except that, in
control 1, nucleic acid molecules that do not bind to
.alpha.-amylase with their 5' ends modified with biotin were used
(the control nucleic acid molecules 1) and, in control 2, only
.alpha.-amylase was used.
[0264] The results obtained are shown in FIG. 11. FIG. 11 is a
photograph showing the results of the pull-down assay using the
bound beads A1 and A5. In FIG. 11, the numerical values on the left
side of the photograph indicate molecular weights, and the
respective lanes show, from the left, the results obtained
regarding the molecular weight marker (M), the bound beads A1 (1),
the bound beads A5 (2), control 1 (C1), and control 2 (AMY). As can
be seen in FIG. 11, in the lane showing the result of control 1, no
band was observed at the same position (about 50 kDa) as in the
lane showing the result of control 2, whereas, in the lanes showing
the results regarding the bound beads A1 and A5, bands were
observed at the same electrophoretic mobility position as in the
lane showing the result of control 2. In other words, the binding
of the .alpha.-amylase-binding nucleic acid molecules 1 and 5 to
.alpha.-amylase was observed. From these results, it was found that
the .alpha.-amylase-binding nucleic acid molecules 1 and 5 bind to
.alpha.-amylase.
Example 5
[0265] The present example examined whether binding nucleic acid
molecules that bind to human LDHS and binding nucleic acid
molecules that bind to human IL-6 can be obtained using MK4.
[0266] (1) Binding Nucleic Acid Molecule
[0267] Binding nucleic acid molecules that bind to human LDHS and
binding nucleic acid molecules that bind to human IL-6 were
obtained in the same manner as in the item (1) in Example 2, except
that, instead of sIgA as the target, human LDHS (manufactured by
Meridian Life Science, Inc., Catalog number: #A38558H-100) and
human IL-6 (manufactured by MP Biomedicals, LLC-Cappel Products,
Catalog number: #55905) were used, respectively.
[0268] As a result, as the binding nucleic acid molecules that bind
to LDHS, binding nucleic acid molecules consisting of the base
sequences of SEQ ID NOs: 17 to 20 shown below were obtained, and as
the binding nucleic acid molecules that bind to IL-6, binding
nucleic acid molecules consisting of the base sequences of SEQ ID
NOs: 21 and 22 shown below were obtained. In the base sequences of
SEQ ID NOs: 17 to 22 shown below, each underlined A is MK4.
TABLE-US-00012 LDH5-binding nucleic acid molecule 1 (SEQ ID NO: 17)
5'-GGAATTGACACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGA
TATCAGGTCTCCTAAGGCTGGCTGGCTACTATAC-3' LDH5-binding nucleic acid
molecule 2 (SEQ ID NO: 18)
5'-GGAATTGACACCTCGCCGTTTATGAGAGGGAGATCATCTCTCTG
GCGGACACAACCTAAGGCTGGCTGGCTACTATAC-3' LDH5-binding nucleic acid
molecule 3 (SEQ ID NO: 19)
5'-ACCTCGCCGTTTATGCTGCTGGCTCGTGAGACGGATATCAGGTC TCCTAAGGCTGGC-3'
LDH5-binding nucleic acid molecule 4 (SEQ ID NO: 20)
5'-TGCTGCTGGCTCGTGAGACGGATATCAGGTCTCCTAAGGCTGG C-3' IL-6-binding
nucleic acid molecule 1 (SEQ ID NO: 21)
5'-GGAATTGACACCTCGCCGTTTATGAGTTCAATGGTATTGTATC
GACTCTTCTCGCCTAAGGCTGGCTGGCTACTATAC-3' IL-6-binding nucleic acid
molecule 2 (SEQ ID NO: 22)
5'-ACCTCGCCGTTTATGAGTTCAATGGTATTGTATCGACTCTTCT C-3'
[0269] (2) Examination of binding by SPR
[0270] The binding between the LDH5-binding nucleic acid molecules
and LDH5 and the binding between the IL-6-binding nucleic acid
molecules and IL-6 were measured in the same manner as in the item
(2) in Example 2, except that the LDH5-binding nucleic acid
molecules or the IL-6-binding nucleic acid molecules having a
20-mer poly(dT) added to their 3' ends were used as the ligand 2
and each of the following proteins was used as the analyte. As
controls, the binding was examined in the same manner except that
.alpha.-amylase and sIgA were used as the analytes. [0271] LDH5:
Lactate Dehydrogenase 5, Human (manufactured by Meridian Life
Science, Inc., Catalogue number: #A38558H-100) [0272] CgA:
Recombinant full length Human Chromogranin A (manufactured by
Creative BioMart, Catalogue number: #CHGA 26904TH) [0273] IL-6:
IL-6, manufactured by Human, Recombinant (manufactured by
PeproTech, Catalogue number: #200-06) [0274] Amylase:
.alpha.-amylase (manufactured by Lee Biosolutions, Catalogue
number: #120-10) [0275] sIgA: IgA (Secretory), Human (manufactured
by MP Biomedicals, LLC-Cappel Products, Catalogue number:
#55905)
[0276] The results of measuring the binding between the respective
types of the LDH5-binding nucleic acid molecule and LDH5 are shown
in FIG. 12, and the results of measuring the binding between the
respective types of IL-6-binding nucleic acid molecules and IL-6
are shown in FIG. 13.
[0277] FIGS. 12A to 12D are graphs showing the binding ability of
the respective types of the LDH5-binding nucleic acid molecules to
LDH5. FIGS. 12A to 12D show the results obtained regarding the
LDH5-binding nucleic acid molecules 1 to 4, respectively. In each
of FIGS. 12A to 12D, the horizontal axis indicates the time elapsed
after the injection of the ligand, and the vertical axis indicates
the relative value (RU) of the binding force. As can be seen in
FIGS. 12A to 12D, the respective types of the LDH5-binding nucleic
acid molecules did not bind to the amylase or sIgA and bound to
LDH5.
[0278] FIGS. 13A and 13B are graphs showing the binding ability of
the respective types of IL-6-binding nucleic acid molecules to
IL-6. FIGS. 13A and 13B show the results obtained regarding the
IL-6-binding nucleic acid molecules 1 and 2, respectively. In each
of FIGS. 13A and 13B, the horizontal axis indicates the time
elapsed after the injection of the ligand, and the vertical axis
indicates the relative value (RU) of the binding force. As can be
seen in FIGS. 13A and 13B, the respective types of IL-6-binding
nucleic acid molecules did not bind to the amylase or sIgA and
bound to IL-6.
[0279] Next, the binding ability of each type of the binding
nucleic acid molecules was examined on the basis of the amount of
each type of the binding nucleic acid molecules immobilized on a
measurement chip and the binding amount of each type of the binding
nucleic acid molecules to the target. Specifically, the signal
intensity (RU) after the injection of the ligand 2 was measured,
and the measured value, which corresponds to a signal indicating
the amount of each type of the binding nucleic acid molecules
immobilized on the measurement chip, was regarded as an "nucleic
acid molecule immobilization measured value (A)". Further, signal
intensity measurement was performed concurrently with injection of
the analyte and washing with a buffer. With 0 seconds being the
start of the injection, the mean value of signal intensities from
115 seconds to 125 seconds was determined. This result, which
corresponds to a signal indicating the binding amount between each
type of the binding nucleic acid molecules and the target, was
regarded as a "target binding measured value (B)". Then, the value
(B/A) obtained by dividing the target binding measured value (B) by
the nucleic acid molecule immobilization measured value (A) was
determined as a relative value (Relative Unit), and the
thus-obtained value was regarded as the binding ability. As
controls, the binding ability was determined in the same manner,
except that .alpha.-amylase and sIgA were used as the analytes.
[0280] The results obtained regarding the respective types of the
LDH5-binding nucleic acid molecules are shown in FIG. 14, and the
results obtained regarding the respective types of IL-6-binding
nucleic acid molecules are shown in FIG. 15.
[0281] FIG. 14 is a graph showing the relative values (Relative
Units) of the binding amounts of the respective types of the
LDH5-binding nucleic acid molecules to LDH5. In FIG. 14, the
horizontal axis indicates the type of the LDH5-binding nucleic acid
molecules, and the vertical axis indicates the relative value. As
can be seen in FIG. 14, no binding was observed in the control in
which .alpha.-amylase or sIgA was used. In contrast, all the types
of the LDH5-binding nucleic acid molecules bound to LDH5.
[0282] FIG. 15 is a graph showing the relative values of the
binding amounts of the respective types of the IL-6-binding nucleic
acid molecules to IL-6. In FIG. 15, the horizontal axis indicates
the types of the IL-6-binding nucleic acid molecules, and the
vertical axis indicates the relative value. As can be seen in FIG.
15, no binding was observed in the control in which .alpha.-amylase
or sIgA was used. In contrast, all the types of the IL-6-binding
nucleic acid molecules bound to IL-6.
[0283] From these results, it was found that a binding nucleic acid
molecule that binds to LDH5 and a binding nucleic acid molecule
that binds to IL-6 can be obtained using MK4, which is the
nucleoside derivative according to the present invention.
[0284] (3) Examination of Binding Force
[0285] The relative value (RU) of the binding force was measured in
the same manner as in the item (2) in Example 5, except that each
type of the LDH5-binding nucleic acid molecules having a 20-mer
poly(T) added to their 3' ends were used as the ligand 2 and that
the concentration of LDH5 as the analyte was set to 1.25, 2.5, 5,
10, or 20 nmol/L or to 6.25, 12.5, 25, 50, or 100 nmol/L. Also, the
relative value of the binding force (RU) was measured in the same
manner as in the item (2) in Example 5, except that each type of
the IL-6-binding nucleic acid molecules having a 20-mer poly(T)
added to their 3' ends were used as the ligand 2 and that the
concentration of IL-6 as the analyte was set to 6.25, 12.5, 25, 50,
or 100 nmol/L or to 12.5, 25, 50, 100, or 200 nmol/L. Then, on the
basis of the relative values of the binding force (RU) measured in
the above, the dissociation constant between each type of the
LDH5-binding nucleic acid molecules and LDH5 and the dissociation
constant between each type of the IL-6-binding nucleic acid
molecules and IL-6 were calculated. The results obtained are shown
in Table 2 below.
TABLE-US-00013 TABLE 2 Dissociation Nucleic acid molecule name
constant (nM) LDH5-binding nucleic acid molecule 1 1.68
LDH5-binding nucleic acid molecule 2 0.49 LDH5-binding nucleic acid
molecule 3 0.53 LDH5-binding nucleic acid molecule 4 0.23
LDH5-binding nucleic acid molecule 5 2.54 LDH5-binding nucleic acid
molecule 6 1.38
[0286] As can be seen in Table 2 above, it was found that these
binding nucleic acid molecules all have excellent binding ability
to the targets. In particular, the LDH5-binding nucleic acid
molecules 2 to 4 exhibited particularly excellent binding ability
to the target.
[0287] (4) Examination of Binding by Pull-Down Assay
[0288] Beads carrying LDH5-binding nucleic acid molecules bound
thereto (also referred to as "bound beads L" hereinafter) were
prepared by bringing the LDH5-binding nucleic acid molecules 1 with
their 5' ends modified with biotin into contact with the
above-described streptavidin-modified beads. Next, SDS-PAGE was
performed and gel was imaged in the same manner as in the item (5)
in Example 2, except that the bound beads L were mixed with a SB
buffer containing 90 (v/v)% saliva (saliva sample) or a SB buffer
containing LDH5 (target sample). Further, as control 1 and control
2, imaging was performed in the same manner except that, in control
1, the following control nucleic acid molecules 3 with their 5'
ends modified with biotin were used and, in control 2, only LDH5
was used.
TABLE-US-00014 Control nucleic acid molecule 3 (SEQ ID NO: 23)
5'-GGAATTGACACCTCGCCGTTTATG-3'
[0289] Next, the results obtained when the bound beads L were used
are shown in FIG. 16. FIG. 16 is a photograph showing the results
of the pull-down assay using the bound beads L. In FIG. 16, the
numerical values on the left side of the photograph indicate
molecular weights, and the respective lanes are, from the left,
lane M (marker), lane 1 (target sample), lane C1 (control 1), lane
LDH5 (control 2), lane M (marker), lane 2 (saliva sample), and lane
C2 (control 1). As can be seen in FIG. 16, in the lane showing the
result of control 1, no band was observed at the same position
(about 35 kDa) as in the lane showing the result of control 2,
whereas, in the lanes showing the results obtained when the target
sample and the saliva sample were used, bands were observed at the
same electrophoretic mobility position as in the lane showing the
result of control 2, as indicated with the arrows in FIG. 16. In
other words, binding of the LDH5-binding nucleic acid molecules 1
to LDH5 was observed. From these results, it was found that the
LDH5-binding nucleic acid molecules bind to LDH5.
[0290] Although the present invention is described above with
reference to embodiments and examples, the present invention is not
limited thereto. Various modifications can be made within the scope
of the present invention which can be understood by those skilled
in the art.
[0291] The present application is based upon and claims the benefit
of priority from Japanese patent application No. 2016-180894, filed
on September 15, 2016 and International patent application No.
PCT/JP2017/020065, filed on May 30, 2017, and the entire disclosure
of which is incorporated herein in its entirety by reference.
[0292] (Supplementary Notes)
[0293] Some or all of the above-described embodiments and examples
may be described, but are not limited to, as the following
Supplementary Notes.
(Supplementary Note 1)
[0294] A nucleoside derivative or a salt thereof, represented by
the following chemical formula (1):
##STR00015##
where in the chemical formula (1),
[0295] Su is an atomic group having a sugar skeleton at a
nucleoside residue or an atomic group having a sugar phosphate
skeleton at a nucleotide residue, and may or may not have a
protecting group,
[0296] L.sup.1 and L.sup.2 are each independently a straight-chain
or branched, saturated or unsaturated hydrocarbon group having 2 to
10 carbon atoms,
[0297] X.sup.1 and X.sup.2 are each independently an imino group
(--NR.sup.1--), an ether group (--O--), or a thioether group
(--S--), and
[0298] the R.sup.1 is a hydrogen atom or a straight-chain or
branched, saturated or unsaturated hydrocarbon group having 2 to 10
carbon atoms.
(Supplementary Note 2)
[0299] The nucleoside derivative or a salt thereof according to
Supplementary Note 1, wherein the X.sup.1 is an imino group
(--NR.sup.1--).
(Supplementary Note 3)
[0300] The nucleoside derivative or a salt thereof according to
Supplementary Note 1 or 2, wherein the X.sup.2 is an imino group
(--NR.sup.1--).
(Supplementary Note 4)
[0301] The nucleoside derivative or a salt thereof according to
Supplementary Note 2 or 3, wherein the R.sup.1 is a hydrogen
atom.
(Supplementary Note 5)
[0302] The nucleoside derivative or a salt thereof according to any
one of Supplementary Notes 1 to 4, wherein the L.sup.1 is a
vinylene group (--CH.dbd.CH--).
(Supplementary Note 6) The nucleoside derivative or a salt thereof
according to any one of Supplementary Notes 1 to 5, wherein the
L.sup.2 is an ethylene group (--CH.sub.2--CH.sub.2--).
(Supplementary Note 7)
[0303] The nucleoside derivative or a salt thereof according to any
one of Supplementary Notes 1 to 6, wherein an atomic group having a
sugar skeleton at the nucleoside residue or an atomic group having
a sugar phosphate skeleton at the nucleotide residue is represented
by the following chemical formula (2):
##STR00016##
where in the chemical formula (2),
[0304] R.sup.2 is a hydrogen atom, a protecting group, or a group
represented by the following chemical formula (3),
[0305] R.sup.3 is a hydrogen atom, a protecting group, or a
phosphoramidite group,
[0306] R.sup.4 is a hydrogen atom, a fluorine atom, a hydroxyl
group, an amino group, or a mercapto group,
##STR00017##
where in the chemical formula (3),
[0307] Y is an oxygen atom or a sulfur atom,
[0308] Z is a hydroxyl group or an imidazole group, and
[0309] m is an integer of 1 to 10.
(Supplementary Note 8)
[0310] The nucleoside derivative or a salt thereof according to any
one of Supplementary Notes 1 to 7, wherein the nucleoside
derivative represented by the chemical formula (1) is a nucleoside
derivative represented by the following chemical formula (4):
##STR00018##
(Supplementary Note 9)
[0311] A polynucleotide synthesis reagent comprising a nucleotide
derivative or a salt thereof that comprises the nucleoside
derivative or a salt thereof according to any one of Supplementary
Notes 1 to 8.
(Supplementary Note 10)
[0312] A method for producing a polynucleotide, comprising the step
of synthesizing a polynucleotide using a nucleotide derivative or a
salt thereof that comprises the nucleoside derivative or a salt
thereof according to any one of Supplementary Notes 1 to 8.
(Supplementary Note 11)
[0313] A polynucleotide comprising, as a building block, a
nucleotide derivative or a salt thereof that comprises the
nucleoside derivative or a salt thereof according to any one of
[0314] Supplementary Notes 1 to 8.
(Supplementary Note 12)
[0315] The polynucleotide according to Supplementary Note 11,
wherein the polynucleotide is a binding nucleic acid molecule that
binds to a target.
(Supplementary Note 13)
[0316] The polynucleotide according to Supplementary Note 12,
wherein the target is at least one selected from the group
consisting of secretory immunoglobulin A, amylase, .beta.-defensin
4A, lysozyme, lactate dehydrogenase (LDH) 5, and interleukin
(IL)-6.
(Supplementary Note 14)
[0317] A method for producing a binding nucleic acid molecule,
comprising the steps of:
[0318] causing a candidate polynucleotide and a target to come into
contact with each other; and
[0319] selecting the candidate polynucleotide bound to the target
as a binding nucleic acid molecule that binds to the target,
wherein
[0320] the candidate polynucleotide is the polynucleotide according
to any one of
[0321] Supplementary Notes 11 to 13.
(Supplementary Note 15)
[0322] The method according to Supplementary Note 14, wherein the
target is at least one selected from the group consisting of
secretory immunoglobulin A, amylase, .beta.-defensin 4A, lysozyme,
lactate dehydrogenase (LDH) 5, and interleukin (IL)-6.
(Supplementary Note 16)
[0323] An .alpha.-amylase-binding nucleic acid molecule comprising
a polynucleotide (a):
[0324] (a) a polynucleotide (a1):
[0325] (a1) a polynucleotide consisting of any of base sequences of
SEQ ID NOs: 1 and 11 to 16.
(Supplementary Note 17)
[0326] The .alpha.-amylase-binding nucleic acid molecule according
to Supplementary Note 16, wherein the .alpha.-amylase-binding
nucleic acid molecule comprises a modified base, which is a base
modified.
(Supplementary Note 18)
[0327] The .alpha.-amylase-binding nucleic acid molecule according
to Supplementary Note 17, wherein the modified base is a modified
purine base, which is a purine base modified with a modifying
group.
(Supplementary Note 19)
[0328] The .alpha.-amylase-binding nucleic acid molecule according
to Supplementary Note 18, wherein modifying group is an adenine
residue.
(Supplementary Note 20)
[0329] The .alpha.-amylase-binding nucleic acid molecule according
to any one of Supplementary Notes 16 to 19, wherein the
polynucleotide is DNA.
(Supplementary Note 21)
[0330] A method for analyzing .alpha.-amylase, comprising the step
of:
[0331] causing a specimen and a nucleic acid molecule to come into
contact with each other to detect .alpha.-amylase in a specimen,
wherein
[0332] the nucleic acid molecule is an .alpha.-amylase-binding
nucleic acid molecule according to any one of Supplementary Notes
16 to 20, and
[0333] in the detection, the nucleic acid molecule is caused to
bind to the .alpha.-amylase in the specimen, and the
.alpha.-amylase in the specimen is detected by detecting the
binding.
(Supplementary Note 22)
[0334] The method according to Supplementary Note 21, wherein the
specimen is at least one selected from the group consisting of
saliva, urine, plasma, and serum.
(Supplementary Note 23)
[0335] A .beta.-defensin (BDN)4A-binding nucleic acid molecule
comprising a polynucleotide (b):
[0336] (b) a polynucleotide (b1):
[0337] (b1) a polynucleotide consisting of any of base sequences of
SEQ ID NOs: 4 to 6.
(Supplementary Note 24)
[0338] The BDN4A-binding nucleic acid molecule according to
Supplementary Note 23, wherein the BDN4A-binding nucleic acid
molecule comprises a modified base, which is a base modified.
(Supplementary Note 25)
[0339] The BDN4A-binding nucleic acid molecule according to
Supplementary Note 24, wherein the modified base is a modified
purine base, which is a purine base modified with a modifying
group.
(Supplementary Note 26)
[0340] The BDN4A-binding nucleic acid molecule according to
Supplementary Note 25, wherein the modifying group is an adenine
residue.
(Supplementary Note 27)
[0341] The BDN4A-binding nucleic acid molecule according to any one
of Supplementary Notes 23 to 26, wherein the polynucleotide is
DNA.
(Supplementary Note 28)
[0342] A method for analyzing BDN4A, comprising the step of:
causing a specimen and a nucleic acid molecule to come into contact
with each other to detect .beta.-defensin (BDN)4A in the specimen,
wherein
[0343] the nucleic acid molecule is the BDN4A binding nucleic acid
molecule according to any one of Supplementary Notes 23 to 27,
and
[0344] in the detection step, the nucleic acid molecule is caused
to bind to the BDN4A in the specimen, and the BDN4A in the specimen
is detected by detecting the binding.
(Supplementary Note 29)
[0345] The method according to Supplementary Note 28, wherein the
specimen is at least one selected from the group consisting of
saliva, urine, plasma, and serum.
(Supplementary Note 30)
[0346] A lysozyme-binding nucleic acid molecule comprising a
polynucleotide (l):
[0347] (l) a polynucleotide (l1):
[0348] (l1) a polynucleotide consisting of any of base sequences of
SEQ ID NOs: 7 to 9.
(Supplementary Note 31)
[0349] The lysozyme-binding nucleic acid molecule according to
Supplementary Note 30, wherein the lysozyme-binding nucleic acid
molecule comprises a modified base, which is a base modified.
(Supplementary Note 32)
[0350] The lysozyme-binding nucleic acid molecule according to
Supplementary Note 31, wherein the modified base is a modified
purine base, which is a purine base modified with a modifying
group.
(Supplementary Note 33)
[0351] The lysozyme-binding nucleic acid molecule according to
Supplementary Note 32, wherein the modifying group is an adenine
residue.
(Supplementary Note 34)
[0352] The lysozyme-binding nucleic acid molecule according to any
one of Supplementary Notes 30 to 33, wherein the polynucleotide is
DNA.
(Supplementary Note 35)
[0353] A method for analyzing lysozyme, comprising the step of:
[0354] causing a specimen and a nucleic acid molecule to come into
contact with each other to detect lysozyme in the specimen,
wherein
[0355] the nucleic acid molecule is the lysozyme-binding nucleic
acid molecule according to any one of Supplementary Notes 30 to 34,
and
[0356] in the detection, the nucleic acid molecule is caused to
bind to the lysozyme in the specimen, and the lysozyme in the
specimen is detected by detecting the binding.
(Supplementary Note 36) The method according to Supplementary Note
35, wherein the specimen is at least one selected from the group
consisting of saliva, urine, plasma, and serum.
(Supplementary Note 37)
[0357] A lactate dehydrogenase (LDH)5-binding nucleic acid molecule
comprising a polynucleotide (d):
[0358] (d) a polynucleotide (d1):
[0359] (d1) a polynucleotide consisting of any of base sequences of
SEQ ID NOs: 17 to 20.
(Supplementary Note 38)
[0360] The LDHS-binding nucleic acid molecule according to
Supplementary Note 37, wherein the LDHS-binding nucleic acid
molecule comprises a modified base, which is a base modified.
(Supplementary Note 39)
[0361] The LDHS-binding nucleic acid molecule according to
Supplementary Note 38, wherein the modified base is a modified
thymine, which is a thymine base modified with a modifying
group.
(Supplementary Note 40)
[0362] The LDHS-binding nucleic acid molecule according to
Supplementary Note 39, wherein the modifying group is an adenine
residue.
(Supplementary Note 41)
[0363] The LDHS-binding nucleic acid molecule according to any one
of Supplementary Notes 37 to 40, wherein the polynucleotide is
DNA.
(Supplementary Note 42)
[0364] A method for analyzing LDH5, comprising the step of:
[0365] causing a specimen and a nucleic acid molecule to come into
contact with each other to detect lactate dehydrogenase (LDH)5 in
the specimen, wherein
[0366] the nucleic acid molecule is the LDH5-binding nucleic acid
molecule according to any one of Supplementary Notes 37 to 41,
and
[0367] in the detection step, the nucleic acid molecule is caused
to bind to the LDH5 in the specimen, and the LDH5 in the specimen
is detected by detecting the binding.
(Supplementary Note 43)
[0368] The method according to Supplementary Note 42, wherein the
specimen is at least one selected from the group consisting of
saliva, urine, plasma, and serum.
(Supplementary Note 44)
[0369] An interleukin (IL)-6 binding nucleic acid molecule
comprising a polynucleotide (i):
[0370] (i) a polynucleotide (i1):
[0371] (i1) a polynucleotide consisting of any of base sequences of
SEQ ID NOs: 21 and 22.
(Supplementary Note 45)
[0372] The IL-6-binding nucleic acid molecule according to
Supplementary Note 44, wherein the IL-6-binding nucleic acid
molecule comprises a modified base, which is a base modified.
(Supplementary Note 46)
[0373] The IL-6-binding nucleic acid molecule according to
Supplementary Note 45, wherein the modified base is a modified
thymine, which is a thymine base modified with a modifying
group.
(Supplementary Note 47)
[0374] The IL-6-binding nucleic acid molecule according to
Supplementary Note 46, wherein the modifying group is an adenine
residue.
(Supplementary Note 48)
[0375] The IL-6-binding nucleic acid molecule according to any one
of Supplementary Notes 44 to 47, wherein the polynucleotide is
DNA.
(Supplementary Note 49)
[0376] A method for analyzing IL-6, comprising the step of:
[0377] causing a specimen and a nucleic acid molecule to come into
contact with each other to detect interleukin (IL)-6 in the
specimen, wherein
[0378] the nucleic acid molecule is the IL-6-binding nucleic acid
molecule according to any one of Supplementary Notes 44 to 48, and
in the detection step, the nucleic acid molecule is caused to bind
to the IL-6 in the specimen, and the IL-6 in the specimen is
detected by detecting the binding.
(Supplementary Note 50)
[0379] The method according to Supplementary Note 49, wherein the
specimen is at least one selected from the group consisting of
saliva, urine, plasma, and serum.
INDUSTRIAL APPLICABILITY
[0380] The present invention can provide a novel nucleoside
derivative or a salt thereof. Further, the nucleoside derivative of
the present invention has two purine ring-like structures. The
nucleoside derivative of the present invention thus has, for
example, a relatively larger number of atoms capable of interacting
within or between molecules than a nucleoside derivative having one
purine ring-like structure. The binding nucleic acid molecule
including the nucleoside derivative of the present invention
therefore has an improved binding ability to a target, for example,
compared to a nucleoside derivative having one purine ring-like
structure. Thus, with the nucleoside derivative of the present
invention, a binding nucleic acid molecule that exhibits excellent
binding ability to a target can be produced, for example.
Accordingly, the present invention is really useful, for example,
in the fields of analysis, medicine, life science, and the like.
[0381] [Sequence Listing] TF16066WO2_ST25.txt
Sequence CWU 1
1
23178DNAArtificial SequenceSynthesized polynucleotide (alpha
amylase binding nucleic acid molecule 1) 1ggtttggacg caatctccct
aatctagtga cgaaaatgta cgagggggtc atttgaaact 60acaatgggcg ggcttatc
78280DNAArtificial SequenceSynthesized polynucleotide (sIgA binding
nucleic acid molecule) 2ggtttggacg caatctccct aatcaagcca cggagagtcc
gaggtgacca ttaagcagga 60aactacaatg ggcgggctta 80323DNAArtificial
SequenceSynthesized polynucleotide (control nucleic acid molecule)
3ggatacctta acgccgccta ttg 23476DNAArtificial SequenceSynthesized
polynucleotide (BDN4A binding nucleic acid molecule 1) 4ggttacacga
gccgcacatt tctattttta cggggtatag ttctctgagg aggagttccc 60aggcgaagtt
gttatc 76569DNAArtificial SequenceSynthesized polynucleotide (BDN4A
binding nucleic acid molecule 2) 5cgagccgcac atttctattt ttacggggta
tagttctctg aggaggagtt cccaggcgaa 60gttgttatc 69676DNAArtificial
SequenceSynthesized polynucleotide (BDN4A binding nucleic acid
molecule 3) 6ggttacacga gccgcacatt tcaccgtgat agttctctga ggaggacttc
tagagttccc 60aggcgaagtt gttatc 76776DNAArtificial
SequenceSynthesized polynucleotide (Lysozyme binding nucleic acid
molecule 1) 7ggttacacga gccgcacatt tctaacggga acttcaaccc atacagtctt
ttgagttccc 60aggcgaagtt gttatc 76853DNAArtificial
SequenceSynthesized polynucleotide (Lysozyme binding nucleic acid
molecule 2) 8cgagccgcac atttctaacg ggaacttcaa cccatacagt cttttgagtt
ccc 53976DNAArtificial SequenceSynthesized polynucleotide (Lysozyme
binding nucleic acid molecule 3) 9ggttacacga gccgcacatt tctttactcc
ggaacccata cagtcttttc cggagttccc 60aggcgaagtt gttatc
761023DNAArtificial SequenceSynthesized polynucleotide (control
nucleic acid molecule 2) 10ggtaaccgcc ctgtcttgat aac
231161DNAArtificial SequenceSynthesized polynucleotide (alpha
amylase binding nucleic acid molecule 2) 11ggtttggacg caatctccct
aatctagtga cgaaaatgta cgagggggtc atttgaaact 60a 611252DNAArtificial
SequenceSynthesized polynucleotide (alpha amylase binding nucleic
acid molecule 3) 12gcaatctccc taatctagtg acgaaaatgt acgagggggt
catttgaaac ta 521378DNAArtificial SequenceSynthesized
polynucleotide (alpha amylase binding nucleic acid molecule 4)
13ggtttggacg caatctccct aatcagacta ttatttcaag tacgtggggg tcttgaaact
60acaatgggcg ggcttatc 781478DNAArtificial SequenceSynthesized
polynucleotide (alpha amylase binding nucleic acid molecule 5)
14ggtttggacg caatctccct aatctaaagt ttctaaacga tgtggcggca ttcagaaact
60acaatgggcg ggcttatc 781560DNAArtificial SequenceSynthesized
polynucleotide (alpha amylase binding nucleic acid molecule 6)
15ggtttggacg caatctccct aatctaaagt ttctaaacga tgtggcggca ttcagaaact
601651DNAArtificial SequenceSynthesized polynucleotide (alpha
amylase binding nucleic acid molecule 7) 16gcaatctccc taatctaaag
tttctaaacg atgtggcggc attcagaaac t 511778DNAArtificial
SequenceSynthesized polynucleotide (LDH5 binding nucleic acid
molecule 1) 17ggaattgaca cctcgccgtt tatgctgctg gctcgtgaga
cggatatcag gtctcctaag 60gctggctggc tactatac 781878DNAArtificial
SequenceSynthesized polynucleotide (LDH5 binding nucleic acid
molecule 2) 18ggaattgaca cctcgccgtt tatgagaggg agatcatctc
tctggcggac acaacctaag 60gctggctggc tactatac 781957DNAArtificial
SequenceSynthesized polynucleotide (LDH5 binding nucleic acid
molecule 3) 19acctcgccgt ttatgctgct ggctcgtgag acggatatca
ggtctcctaa ggctggc 572044DNAArtificial SequenceSynthesized
polynucleotide (LDH5 binding nucleic acid molecule 4) 20tgctgctggc
tcgtgagacg gatatcaggt ctcctaaggc tggc 442178DNAArtificial
SequenceSynthesized polynucleotide (IL-6 binding nucleic acid
molecule 1) 21ggaattgaca cctcgccgtt tatgagttca atggtattgt
atcgactctt ctcgcctaag 60gctggctggc tactatac 782244DNAArtificial
SequenceSynthesized polynucleotide (IL-6 binding nucleic acid
molecule 2) 22acctcgccgt ttatgagttc aatggtattg tatcgactct tctc
442324DNAArtificial SequenceSynthesized polynucleotide (control
nucleic acid molecule 3) 23ggaattgaca cctcgccgtt tatg 24
* * * * *
References